Adrenal fibroblasts from adult rats acquire some adrenocortical parenchymal characteristics as a consequence of transformation in early passage with Kirsten murine sarcoma virus. To further define the effects of Kirsten murine sarcoma virus-induced transformation on the steroid enzymes of these cells, we investigated the capacity of Kirsten murine sarcoma virus-transformed and untransformed adrenocortical fibroblasts to convert progesterone to C19 and C21 steroid metabolites. Over 95% of metabolites produced were identified and quantitated, and rates of enzyme activities over 24 h were calculated. The transformed and untransformed cells exhibited 5 alpha- and 5 beta-reductase, 3 alpha-, 3 beta-, and 20 alpha-hydroxysteroid dehydrogenase; 11 beta-, 17-, and 21-hydroxylase (HY); and C17-20-lyase activities. Viral transformation resulted in several metabolites not found in untransformed cells, significantly increased 5 beta-reductase, 3 beta-hydroxysteroid dehydrogenase, and C17-20-lyase activities, and significantly decreased 5 alpha-reductase, 3 alpha- and 20 alpha-hydroxysteroid dehydrogenase, and 21-HY activities. The 11 beta-HY and 17-HY activities remained unchanged. The results support previous data suggesting that adrenocortical fibroblasts express some characteristics of adrenocortical parenchymal stem cells. In contrast to other experimental systems, viral transformation of adrenocortical fibroblasts did not cause a generalized reduction of differentiated functions. Instead, specific increases and decreases in individual enzyme activities, with persisting synthesis of fetal and adult adrenocortico-specific steroids, resulted in an altered steroid profile that may have unique effects on the biology of the malignant cells.