The differences in intracellular and extracellular protein expressions between human prostate cancer fines LNCap and DU145 were examined. The proteins of the two cell lines were extracted and condensed by using protein extraction kits. And the intracellular and extracellular proteins were quantitatively detected on a micro-plate reader by using bicinchoninie acid (BCA) method. The proteins in cell culture fluid were qualitatively assayed by SELDI-TOF-MS. The results showed that the intracellular protein contents of LNCap cells were extremely higher than those of DU145 cells. After serum-free culture, both intracellular and extracellular protein contents of LNCap and DU145 were decreased to some extent. And the intracellular proteins were decreased by 5% in LNCap and by 36% in DU145 respectively, while the extracellular proteins were decreased by 89% in LNCap and 96% in DU145 respectively. SELDI assay revealed that there were 5 marker proteins in LNCap and 6 in DU145. Although both LNCap and DU145 cell lines originated from human prostate cancer, they had some differences in protein expression.