Dispersed single cell suspensions of Syrian hamster insulinoma cells were used to study the effects of a variety of sulfhydryl-binding reagents on insulin release and 45Ca++ flux. Incubation of cells with several organic mercurials resulted in a rapid increase in 45Ca++ uptake as well as increased efflux in cells which had been prelabeled with 45Ca++. Concomitant with increased calcium uptake was a 4- to 5-fold increase in insulin released into the medium. Incubation with alkylating reagents such as iodoacetamide and N-ethyl maleimide or dithiol reagents such as 5,5'-dithiobis (2-nitrobenzoic acid) failed to stimulate either 45Ca++ flux or insulin release. Elimination of medium calscium or preincubation of cells with N-ethyl maleimide resulted in approximately 50% inhibition of mercurial-induced insulin release from these cells. 8-(N,N2-diethylamino)Octyl-3,4,5-trimethoxybenzoate or alpha-isopropyl-alpha [(N-methyl-N-homoveratryl)-gamma-aminopropyl]3,4,5'-trimethoxyphenylacetonitrite hydrochloride, agents which block potassium (40 mM)-stimulated calcium flux and insulin release, failed to inhibit mercurial-induced calcium flux or insulin secretion. These results indicate that sulfhydryl-binding reagents, through their interaction with critical thiol groups, promote insulin release in these insulinoma cells by inducing changes in calcium fluxes. It is possible that these thiol groups regulate calcium metabolism and, thus, insulin release under physiological conditions.