Programmed cell death(PCD) plays a critical role in the development of plant pigment glands, while H_2O_2, which is a kind of reactive oxygen species(ROS) produced by the aerobic metabolism of cells, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H_2O_2 in pigment glands of Gossypium hirsutum L. with 3,3-diaminobenzidine(DAB) staining, 2’,7’-dichlorodihydrofluorescein diacetate(DCFH2)-DA fluorescent labeling and Ce Cl3 cytochemical localization techniques. The results showed that the pigment glands of G. hirsutum could generate H_2O_2, and the amount and localization of H_2O_2 varied at different developmental stages. At the early developmental stage, a small amount of H_2O_2 accumulated in the vacuole membrane of pigment gland cells. At the intermediate stage, a large number of H_2O_2 appeared in the vacuole membrane, while cell walls started to accumulate a small amount of H_2O_2. When pigment gland cell degraded, H_2O_2 mainly accumulated on the chloroplast envelope membrane of inner sheath cells. With the degradation of the sheath cells, H_2O_2 was detected in cell wall and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H_2O_2 would disappeared. The accumulation sites of H_2O_2 are consistent with the process of PCD of individual gland cells, which started from the degradation of intracellular membrane and ended with the degradation of cell walls. Thus H_2O_2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H_2O_2 are not associated with the light, and no H_2O_2 was detected in the secretions of pigment glands. Programmed cell death (PCD) plays a critical role in the development of plant pigment glands, while H_2O_2, which is a kind of reactive oxygen species (ROS) produced by the aerobic metabolism of cells, acts as an important signal in this process. Here , we investigated the temporal and spatial dynamics of accumulated H 2 O 2 in pigment glands of Gossypium hirsutum L. with 3,3-diaminobenzidine (DAB) staining, 2 ’, 7’-dichlorodihydrofluorescein diacetate (DCFH2) -DA fluorescent labeling and Ce Cl3 cytochemical localization techniques. The results showed that the pigment glands of G. hirsutum could generate H_2O_2, and the amount and localization of H_2O_2 varied at different developmental stages. At the early developmental stage, a small amount of H_2O_2 accumulated in the vacuole membrane of pigment gland cells . At the intermediate stage, a large number of H 2 O 2 appeared in the vacuole membrane, while cell walls started to accumulate a small amount of H 2 O 2. When pigment gland cell degraded, H _2O_2 mainly accumulated on the chloroplast envelope membrane of inner sheath cells. With the degradation of the sheath cells, H_2O_2 was detected in cell wall and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H_2O_2 The accumulation sites of H_2O_2 are consistent with the process of PCD of individual gland cells, which started from the degradation of intracellular membrane and ended with the degradation of cell walls. Thus H_2O_2 probably plays an important role in the development of pigment glands . In addition, the development of pigment glands and the generation of H_2O_2 are not associated with the light, and no H_2O_2 was detected in the secretions of pigment glands.