Three SV40-transformed derivatives (G1, T2, and T5) of human prostatic epithelial cells were analyzed karyotypically. For comparison, an SV40 derivative (TA) obtained from isogenic fibroblasts, was also studied. The chromosome complements of these cells, as well as a sub-clone of T2 isolated in soft agar (T2-A5), were analyzed using banding techniques. Numerical as well as structural changes were observed in all transformed cultures. Karyotypic changes in all cells at a given passage level appeared to be random. On the other hand, characteristic differences in modal chromosome number, and type and number of abnormal chromosomes were observed among the different lines. Most cells of two of the three epithelial lines (T1 and T2) were either hypo- or pseudodiploid, whereas T5 consisted of a mixed hypodiploid and hypotetraploid population. The TA subline was also predominantly hypo- and pseudodiploid. Dicentrics, telomeric associations, translocations, and loss of chromosomes were the most prominent abnormalities. The loss of chromosome 18 was characteristic for all epithelial lines. All T1 and T5 cells had lost either one or both copies of the 18. While individual cells of the original T2 line had random karyotypes, most of T2-A5 cells had a relatively uniform karyotypic pattern. They also had a similar pattern of abnormal chromosomes. These observations suggest that culture in soft agar may have selected a particular chromosomal variant. We conclude that transformation of prostatic epithelial cells by SV40 may bring about site-specific as well as random chromosomal changes. These changes could reflect either intermediate or sequential stages in progression to neoplasia.