The keratins are a component of the cytoskeleton that is present in fetal and neonatal rat granulosa cells (ROG), but disappears as the cells undergo postnatal steroidogenic differentiation. Steroidogenesis is initiated in the fetus as a low level constitutive function which is cAMP responsive, but becomes responsive to gonadotrophic hormones only after birth. ROG from PMSG-primed immature rats, like mature ROG, are keratin negative, highly steroidogenic and gonadotrophin-responsive, but rapidly lose their steroidogenic capacity in culture. In such cultured cells, transformation with the Kirsten-ras oncogene (v-Ki-ras) maintains low levels of constitutive steroidogenesis and responsiveness to cAMP, and induces the expression of keratin. To determine whether similar changes would occur in cells expressing both the SV40 and v-Ki-ras, cultured ROG were transformed with SV40 early genes, with Kirsten murine sarcoma virus (KiMSV), or with both agents concurrently. Keratin was demonstrated by fluorescence microscopy and Western blots, and progesterone production by RIA. ROG transformed with SV40 alone became immortalized but secreted little steroid and lacked keratin. In contrast, three cell lines, co-transformed with SV40 plus KiMSV, acquired keratin as well as the capacity to secrete progesterone in response to cAMP, closely resembling cells transformed with Ki-ras alone. KiMSV-transformed muscle fascia fibroblasts lacked both steroidogenic potential and keratin. The results show that the complex, v-Ki-ras-induced changes in steroidogenesis and keratin expression are reproducible and tissue specific. The phenotypic resemblance between singly and doubly transformed ROG indicates that the v-Ki-ras oncogene does not act by overcoming SV40-mediated inhibition of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)