The aim of the present study was to evaluate whether HIV protease inhibitors directly interfere with stimulus-secretion coupling in pancreatic beta-cells. Insulin secretion was determined by a radioimmunoassay (RIA), cytosolic free Ca2+ concentration ([Ca2+]c) with the fluorescence dye fura-2 and whole-cell membrane currents with the patch-clamp technique. Glucose-induced insulin secretion was inhibited in a concentration-dependent manner by ritonavir and nelfinavir but not by indinavir. Ritonavir and nelfinavir lowered [Ca2+]c in the presence of a stimulatory glucose concentration whereas indinavir again had no effect. Ritonavir and nelfinavir completely inhibited the effect of tolbutamide, which normally increases [Ca2+]c by blocking KATP channels. This observation points to an action of both drugs on KATP channels or a step distal to these channels in stimulus-secretion coupling. Ritonavir was used to further evaluate the direct effects of HIV protease inhibitors on beta-cell ion channel currents. Unexpectedly, ritonavir inhibited neither the whole-cell KATP current nor the whole-cell L-type Ca2+ current. Tolbutamide almost completely suppressed the KATP current in the presence of ritonavir excluding that ritonavir alters the tolbutamide sensitivity of the KATP channel. Ritonavir increased the length and decreased the frequency of glucose-induced action potentials. This effect can be attributed to inhibition of voltage-dependent K+ currents. Intracellular stores seem not to be involved in the ritonavir-induced lowering of [Ca2+]c. In conclusion, different HIV protease inhibitors surprisingly reveal distinct effects on insulin secretion. Ritonavir inhibits insulin secretion by lowering [Ca2+]c but this effect is evidently independent of the opening of KATP channels or the closure of voltage-dependent Ca2+ channels, which are commonly considered to play a key role in stimulus-secretion coupling.