Insulin and calcium ionophores rapidly stimulated transcription of the cytoskeletal beta- and gamma-actin genes in serum-deprived rat H4-II-E hepatoma cells. The calcium ionophore A23187 (1 microM) stimulated transcription of the beta-actin gene by 7.3-, 5.4-, and 2.6-fold and the gamma-actin gene by 5.9-, 5.6-, and 2.6-fold at 15, 30, and 60 min, respectively. Ionomycin (1 microM) similarly increased beta- and gamma-actin transcription. Insulin stimulated beta-actin transcription 11.4-fold and gamma-actin 8.4-fold at 30 min. alpha-Tubulin transcription was induced by both insulin and calcium ionophores but to a lesser degree. The effects of A23187 or ionomycin together with insulin for 30 min were no greater than those of insulin alone. Insulin alone, however, did not significantly increase measurable intracellular calcium concentrations in fura-2-loaded cells. When cytosolic calcium was chelated using quin2 acetoxymethyl ester, the ability of A23187 to increase beta- and gamma-actin transcription was completely abolished, whereas insulin's ability to stimulate actin transcription was only partially inhibited. This suggests that the regulation of gene transcription by insulin may include calcium-dependent pathways but strongly implies that calcium-independent pathways are also utilized.