Transcription of the c-fos proto-oncogene is rapidly induced in the rat pheochromocytoma PC12 cell line by a wide variety of stimuli, including polypeptide growth factors, phorbol esters, and calcium ion fluxes. We have mapped the upstream sequence requirements for this activation in PC12 cells by analysis of promoter deletion mutants in a transient expression assay. Two distinct pathways of c-fos induction are defined that differ in their requirement for cis-acting DNA sequences. Calcium activation of c-fos transcription is dependent on a DNA element located approximately 60 base pairs upstream of the transcription start site. This region is highly conserved between human, mouse, and chicken c-fos genes and contains a sequence that resembles the consensus for a cyclic AMP response element. The dyad symmetry element at position -300, which is necessary for serum responsiveness of c-fos, appears to be unimportant for calcium activation of the gene. The dyad symmetry element is, however, an essential cis-acting sequence for c-fos inducibility by nerve growth factor, epidermal growth factor, fibroblast growth factor, and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. Studies in vivo and in vitro with various mutants of the dyad symmetry element indicate that c-fos activation by polypeptide growth factors and 12-O-tetradecanoyl activation by polypeptide growth factors and 12-O-tetradecanoyl phorbol-13-acetate is mediated by a common transcription factor, and that this factor is identical to the previously described serum response factor. In vitro DNA-binding assays suggest that the quantity of serum response factor-binding activity remains unchanged during c-fos transcriptional activation.