To test the putative role of c-fos in F9 differentiation, we have attempted to inhibit c-fos expression in these cells using an SV40-based expression vector (pSVneo-sof) that programs expression of c-fos antisense (sof) sequences as a 3' extension of a neo mRNA transcript. Of six G418-resistant clones isolated in transfection experiments, five expressed neo-sof transcripts. Two clones synthesized polyadenylated mRNA of the expected size (3.8 kb), two were smaller than expected, and one was larger. Two clones that expressed reduced levels of c-fos protein were inhibited in the induction of laminin, type IV collagen, and proteoglycan-19 RNA transcripts measured after 4 days of differentiation induction with RA and dibutyryl cyclic AMP. Also inhibited was the induction of the differentiation markers, TROMA-1 and TROMA-3. Antisense-expressing cells were not inhibited in the differentiation pathway to visceral endoderm since the alpha-fetoprotein gene was activated normally. We conclude that c-fos antisense expression inhibits some aspects of differentiation in F9 cells.