Objective To observe the morphological changes in in vitro growth of preantral follicle isolated from prepuberal mice and to assess impacts of gonadotropin (Gn),insulin transferrin selenium (ITS) and epidermal growth factor (EGF) on their development.Methods Early preantral mice follicles (90-130 μm diameter) were mechanical isolated and selected from 2 weeks old mice and then cultured in alpha-minimal essential medium (α-MEM) with or without Gn, ITS and EGF. The preantral follicles were cultured singly in 20 microliters droplets for up to 14 d. The medium was replaced and the follicles were observed everyday. Granulosa cells (GC) prolification, antrum formation and oocyte maturation were recorded.Results The medium with Gn supported preantral follicle culture in vitro, during which they retained a three-dimensional structure, maintained oocytes viability and increased in diameter and number of somatic cells. Preantral follicles cultured in Gn medium grew obviously, while those without Gn grew slowly and after 6 ds culture began to shrink and blacken. Significant increase in survival rate and maturation rate of oocytes was observed in Gn group (P＜0. 01), with 92.9% survived and 28. 7% formed an antrum. Further supplementation of the Gn medium with ITS and rLH, resulted in the significant increase in survival and maturation of preantral follicle (P＜0. 05)Conclusions α-MEM can be the medium for in vitro culture (IVC) of preantral follicles,but need to be added with rLH/rFSH, rHCG/rEGF to facilitate thecal cell attachment,GC proliferation and oocyte maturation.