p53 gene structure and chromosome 17p alleles were studied in the three human prostate cancer cell lines, LNCaP, DU-145, and PC-3. Our laboratory has two separate culture lines of the LNCaP human prostate cancer cells. One strain, LNCaP-GW, had a mutation in one of two alleles at position 273 (arg > his). This mutation could not be detected in a second strain of LNCaP, LNCaP-ATCC. Immunohistochemical staining for P53 protein in the cell lines indicated that protein overexpression in LNCaP was heterogeneous, even in clonal isolates derived from LNCaP-GW that contained the codon 273 mutation in every cell. We also performed in vitro and in vivo growth analysis to compare the LNCaP-GW and LNCaP-ATCC cells. LNCaP-GW grew more rapidly than LNCaP-ATCC in vitro. However, LNCaP-ATCC formed tumors efficiently when inoculated into nude mice, whereas LNCaP-GW formed tumors much less efficiently. Consideration must be given to the notion that some of these p53 mutations arose during in vitro passage. We also confirmed published findings with two other human prostate cancer cell lines. In DU-145, two mutations were found in the p53 gene. A mutation at codon 274 (pro > leu) and a second mutation at codon 223 (val > phe) were present. PC-3 cells were hemizygous for chromosome 17p. The single copy of the p53 gene had a base pair deletion at codon 138 that generated a frame shift and a new in-frame stop codon at position 169.