Among the earliest responses to mitogens that have been detected in normal quiescent cells are ionic changes: we have described rapid increases in the cytosolic free Ca2+ concentration ([Ca]i) and in the intracellular pH (pHi) in mitogen-stimulated thymocytes and fibroblasts (Hesketh, T. R., Moore, J. P., Morris, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A., and Metcalfe, J. C. (1985) Nature 313, 482-484). Here we investigate the relationship between these ionic signals and the subsequent expression of the c-fos and c-myc proto-oncogenes in murine thymocytes. We show that the plant lectin concanavalin A (ConA), the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the Ca2+-ionophore A23187 each causes a rapid increase in both c-fos and c-myc mRNAs. The activation of both genes is completely dependent on the extracellular Ca2+ concentration ([Ca]o) for A23187 and independent of [Ca]o for TPA. Activation of c-myc, but not c-fos, by ConA is partially dependent on [Ca]o. The pHi increases generated by ConA or TPA are not necessary for expression of mRNA from either gene in response to these mitogens. Exogenous 8-bromo-cyclic AMP (but not 8-bromo-cyclic GMP) inhibits the c-myc responses to ConA and TPA. The data also show that neither early c-fos nor c-myc expression is sufficient to commit the cells to DNA synthesis.