Murine 3T3 fibroblasts, treated with mitomycin C in order to inhibit their proliferation, have been extensively used as feeder layers to enhance the cultivation of human keratinocytes in vitro. In order to identify possible factors responsible for this enhancing effect, studies were undertaken to determine whether eicosanoid generation by 3T3 cells was preserved following mitomycin C treatment. Accordingly, both untreated and mitomycin C-treated 3T3 cells were incubated with [1-14C]arachidonic acid and their qualitative generation of [1-14C]labeled eicosanoids assessed by thin layer chromatography. Levels of endogenously generated eicosanoids were quantitatively determined by specific radioimmunoassays performed on culture supernatants obtained from incubations of both treated and untreated cells. Results of these studies demonstrate that mitomycin C treatment, although preventing the proliferation of 3T3 cells, failed to alter their generation of prostaglandin E2 or 6-keto-prostaglandin F1 alpha. Furthermore, in parallel cultures, the untreated 3T3 cells stopped generating eicosanoids upon reaching confluence, whereas the mitomycin C-treated 3T3 cells that did not proliferate continued to generate eicosanoids.