The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.