The c-fos proto-oncogene is rapidly inducible by a variety of extracellular stimuli. In order to dissect the intracellular signalling pathways responsible for c-fos induction, we have used a gel electrophoresis DNA-binding assay to identify trans-acting factors that bind to c-fos regulatory regions. We have identified a factor in Balb/c-3T3 cells that binds to an oligonucleotide, containing sequences -351 to -337 of the human c-fos gene. This factor is inducible in quiescent cells within 30 min of addition of conditioned medium from v-sis transformed cells. Cycloheximide fails to block this induction. This result suggests that the factor is present in an inactive form in quiescent cells and is activated only in response to the appropriate inducer. Fibroblast growth factor (FGF) and the tumor promoter phorbol myristate acetate (TPA) induce the c-fos gene but not this DNA-binding activity. We propose from this that there are multiple regulatory regions upstream of c-fos each capable of responding to a different set of stimuli.