The c-fos proto-oncogene is believed to play a pivotal role in transducing growth factor-mediated signals from the extracellular milieu into the nucleus. c-fos protein dimerizes with c-jun and related proteins and mediates transcription via AP-1 sites. Using c-fos-deficient mice generated through gene knockout techniques, we derived 3T3-type cell lines from primary embryonic fibroblasts. The c-fos-deficient cells grow normally under optimal culture conditions and show only a slight reduction in growth rate in low serum culture compared with control cells. They also express mRNA for most of the Fos and Jun family members at normal levels. The overall levels of AP-1 DNA binding activity are normal and several genes (c-jun, MCP1, metallothionein) known to contain functional AP-1 sites are expressed normally in the c-fos-deficient and control cells. In contrast, mRNA for the metalloproteases stromelysin (MMP-3) and type I collagenase (MMP-1), which are often induced by oncogenes and growth factors and have been implicated in tumor invasiveness, cannot be induced by epidermal growth factor or platelet-derived growth factor in c-fos-deficient cells. Transformation of mutant cells with polyoma middle T oncogene essentially restores wild-type levels of stromelysin expression, while transformation with v-src leads to only a weak induction of the metalloprotease. These results clearly demonstrate that some AP-1-dependent genes require c-fos for full expression while others do not; oncogenes may activate expression of metalloproteases via either fos-dependent or fos-independent mechanisms. These results also imply that c-fos may play an important regulatory role in the invasive behavior of malignant tumors, independent of any role this proto-oncogene might play in cell growth per se.