Both increases in c‐fos proto‐oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet‐derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF‐induced increases in c‐fos proto‐oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose‐response relationships and mechanisms of growth factor‐induced changes in these two parameters as well as DNA synthesis. Concentration‐dependent increases in [Ca2+]i, c‐fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF‐induced increases in c‐fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin‐2, and TMB‐8. Under these conditions, PDGF‐induced DNA synthesis was blocked, whereas c‐fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF‐induced c‐fos expression was inhibited by 75–80%, while PDGF‐induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC‐independent component of PDGF‐stimulated c‐fos expression was found to be independent of increases in [Ca2+]i. These data suggest that (1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c‐fos proto‐oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, (2) PDGF‐stimulated increases in [Ca2+]i are not required for c‐fos expression, and (3) distinct pathways regulate PDGF‐induced c‐fos expression and mitogenesis, with c‐fos expression being substantially PKC‐dependent yet [Ca2+]i‐independent, while mitogenesis is [Ca2+]i‐dependent yet PKC‐independent.