We recently demonstrated the mitogenic and secretagogic actions of lithium in the insulin-producing pancreatic beta-cell (Sjöholm A, Welsh N, Hellerström C: Lithium increases DNA replication, polyamine content and insulin secretion by rat pancreatic beta-cells in vitro. Am J Physiol 262:C391-C395, 1992). In this study, the influence of lithium on beta-cell signal transduction pathways was monitored and their importance for the stimulated cell replication and hormone secretion was elucidated by selective pharmacological probes. To this end, fetal rat pancreatic islets enriched in beta-cells were isolated and cultured for 3 days with or without 10 mmol/l LiCl. This resulted in a marked mitogenic response by the beta-cells, of similar magnitude to that obtained by pharmacological activation of cAMP-dependent protein kinases by forskolin or the Sp-diastereomer of cAMP or protein kinase C stimulation by phorbol ester. However, neither did lithium affect the islet content of cAMP (whereas forskolin did), nor was the mitogenic response to the ion impeded when islets were pretreated with the Rp-diastereomer of cAMP, a specific antagonist of cAMP-dependent protein kinases, or by the Ca2+ channel blocker D-600. The protein kinase C inhibitor 1-(5'-isoquinolinesulfonyl)-2-methylpiperazine prevented the mitogenicity of phorbol ester, but not that of lithium. Conversely, addition of increasing concentrations of inositol along with lithium also did not affect the mitogenicity of the ion, providing indirect evidence against the involvement of protein kinase C in mediating the growth-promoting effect of lithium in this system. It was found that pretreatment of islets with pertussis toxin, which inactivates GTP-binding proteins by ADP-ribosylation, prevented the mitogenicity and part of the secretagogic action of lithium. It is concluded that although specific activation of the cAMP and protein kinase C signaling systems appears sufficient to trigger a mitogenic response of the beta-cell, lithium seemingly does not work through any of these systems nor via Ca2+ influx in promoting beta-cell mitogenesis. Our results, moreover, suggest that the actions of lithium are conveyed by pertussis toxin-sensitive GTP-binding proteins.