Autocrine epidermal growth factor receptor activation by transforming growth factor α (TGFα) has been implicated in growth stimulation during epithelial neoplasia. Using keratinocytes isolated from mice with genetic defects in TGFα expression, we tested whether TGFα is required for transformation by the v-ras^Ha oncogene. Introduction of v-ras^Ha into primary epidermal cultures using a retroviral vector stimulated growth of both control (TGFα +/+, BALB/c) and TGFα-deficient (TGFα -/-, wa-1) keratinocytes. Moreover, v-ras^Ha elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal growth factor (EGF) receptor activation, in both TGFα +/+ and TGFα -/- keratinocytes. v-ras^Ha markedly increased secreted (>10-fold) and cell-associated (2–3-fold) TGFα levels in keratinocytes from TGFα +/+ and BALB/c mice, but not TGFα -/- or wa-1 mice. Based on Northern blot analysis, v-ras^Ha induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like growth factor, and betacellulin in cultured keratinocytes from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGFα transcript, wa-1 keratinocytes expressed two additional TGFα transcripts, 4.7 and 5.2 kilobases long. All three transcripts were up-regulated in response to v-ras^Ha, as well as exogenous TGFα or keratinocyte growth factor treatment, and were also detected in RNA isolated from wa-1 brain and skin. In vivo, v-ras^Ha keratinocytes from control as well as TGFα-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like growth factor, and betacellulin mRNA, regardless of their TGFα status. These findings indicate that TGFα is not essential for epidermal neoplasia induced by the v-ras^Ha oncogene and suggest that another EGF family member(s) may contribute to autocrine growth stimulation of ras-transformed keratinocytes.