The pharmacological properties of alpha-2 adrenoceptors and the existence of nonadrenergic idazoxan-binding sites (NAIBS) were investigated in the insulin-secreting cell-line, RINm5F, using [3H]RX821002 and [3H]idazoxan. Analysis of [3H]RX821002 saturation isotherms revealed the presence of a single class of binding sites (Bmax = 47.5 +/- 3.5 fmol/mg protein) having high affinity (Kd = 1.26 +/- 0.18 nM). Inhibition of [3H]RX821002 binding by adrenergic compounds showed that the labeled sites displayed the properties expected for an alpha-2 adrenoceptor. Based on competition data with drugs having alpha-2 adrenoceptor subtype selectivity, the receptor from RINm5F is neither an alpha-2B nor an alpha-2C. It resembles the alpha-2A, but deviates from this subtype because of a weak affinity for yohimbine and rauwolscine. In this respect, RINm5F alpha-2 adrenoceptor is identical to the receptor previously described in rat intestinal mucosa and corresponds to a fourth subtype: alpha-2D. Agonist inhibition curves were better fitted by a two-site model and indicated that about half of the receptor population was under a high-affinity state corresponding to G protein-coupled receptors. [32P]ADP-ribosylation with pertussis toxin and immunodetection with specific antibodies permitted the identification of three distinct G proteins: Gi2, Gi3 and G0. Binding experiments with [3H]idazoxan showed that this imidazoline labeled two types of sites corresponding to alpha-2 adrenoceptors and NAIBS. Analysis of saturation isotherms under binding conditions allowing to discriminate between the two site populations indicated that the density of NAIBS (44 +/- 2 fmol/mg protein) was fairly identical to that of alpha-2 adrenoceptors. The pharmacological properties of NAIBS, as assessed by determining the relative affinity of imidazolinic and nonimidazolinic compounds, reasonably matched that reported in other tissues. Taken together, these data make the RINm5F cell-line 1) the first model in permanent culture known as expressing an alpha-2 adrenoceptor of the alpha-2D subtype; 2) a good system for studying in vitro the respective role of alpha-2 adrenoceptors and NAIBS in the regulation of insulin secretion by beta cells.