We have previously shown that insulin is less effective in inducing expression of several genes in H4 hepatoma cells with reduced functional protein kinase-C (PKC) activity. However, other reports suggest that insulin regulation of gene transcription is not PKC dependent. Insulin and phorbol 12-myristate 13-acetate (PMA) rapidly inhibit transcription of the tyrosine aminotransferase and albumin genes. Prolonged PMA pretreatment, to desensitize cells to PMA, resulted in a loss of insulin ability to inhibit albumin transcription. Insulin was still able to inhibit tyrosine aminotransferase transcription, but less than in non-PMA-pretreated cells, and there was also a slight decrease in the ability of insulin to inhibit phosphoenolpyruvate carboxykinase transcription. We previously demonstrated decreased responsiveness of PMA-induced gene expression in insulin-desensitized cells. In the present work, using insulin-desensitized H4 cells (insulin pretreatment for 24 h), subsequent treatment with PMA did not alter phosphoenolpyruvate carboxykinase transcription rates, whereas PMA did inhibit tyrosine aminotransferase transcription rates to an extent similar to observed in nonpretreated cells. Unexpectedly, there was a significant increase in albumin transcription after PMA addition to insulin-pretreated cells. These findings support our hypothesis that the role of PKC in the regulation of gene expression by insulin varies for different insulin-regulated genes.