We reported previously that in pancreatic islet cells, certain diacylglycerols (DGs) evoke increases in cytosolic calcium ([Ca2+]i), mainly by intracellular mobilization. We now examined the effects of DGs on the increase in [Ca2+]i due to Ca2+ influx. In the insulin-secreting HIT T-15 islet cell line, cell membrane depolarization using 40 mM KCl evoked a 2- to 3-fold increase in [Ca2+]i, which lasted several minutes. A cell-permeable DG, 1,2-dioctanoylglycerol (DiC8; 10 microM) induced a 12 +/- 4% rise in [Ca2+]i, which did not occur in the absence of extracellular Ca2+ or in the presence of verapamil; this effect was not protein kinase-C (PKC) dependent, because it was not altered by the addition of the PKC inhibitor staurosporine or by using PKC-depleted cells. When DiC8 was added first, the KCl-induced increase in [Ca2+]i was inhibited in a dose-dependent manner (100% at 10-15 microM DiC8); this effect was PKC independent. At a concentration of 10 microM, other synthetic DGs, 1,2-dihexanoylglycerol (DiC6), 1,2-didecanoylglycerol (DiC10), or 1-oleoyl-2-acetylglycerol, inhibited the KCl-induced rise in [Ca2+]i to 15 +/- 4%, 47 +/- 7%, and 51 +/- 5% of the control value, respectively. R59022 (10 microM), which inhibits DG kinase and causes accumulation of endogenous DGs, inhibited the KCl-induced rise in [Ca2+]i to 2 +/- 0.2% of the control value; this inhibition was not affected by staurosporine. In anchored cells, KCl stimulated insulin release (959 +/- 88 microU/mg protein above the control value); 20 microM DiC6 or DiC8 attenuated KCl-induced insulin release by 68% and 31% of the control value, respectively; DiC10 or 1-oleoyl-2-acetylglycerol had no effect. R59022 inhibited KCl-induced insulin release by 90% of the control value. We conclude that in HIT T-15 cells, DGs may serve as positive and negative modulators of [Ca2+]i, apparently by complex and PKC-independent mechanisms. These divergent actions of DGs on islet cell Ca2+ balance together with the accompanying activation of PKC affect insulin release in a complex manner.