We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca 2 + ] i , and IP 3 in β-TC3 cells. Carbachol (0.1-100 μM) increased [Ca 2 + ] i and carbachol (0.1-1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP 3 ) production. U-73122 (2-12 μM) inhibited the effects of carbachol on [Ca 2 + ] i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca 2 + ] i by 20%, and did not inhibit the effect of carbachol on insulin release. Since carbachol increases IP 3 , [Ca 2 + ] i , and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-dependent processes in β-TC3 cells.