Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.