High-affinity binding sites for benzodiazepines are present in a wide range of tissues other than brain, and appear to be pharmacologically distinct from central nervous system receptors. Binding sites have been reported on rat peritoneal mast cells, but specificity of binding and functional effects have not been addressed in these cells. Using the specific radioligand [3H]-Ro5-4864, which binds to peripheral but not central binding sites, we characterized binding to rat peritoneal mast cells and the related cell line, RBL-1 cells, and effects on granule secretion. Studies in rat peritoneal mast cells disclosed one class of saturable binding sites with a dissociation constant of KD = 5.29 +/- 1.26 nM (mean +/- SE) and maximal binding Bmax = 357 +/- 113 fmol/10(6) cells. Competition studies showed that IC50 for Ro5-4864 was 15.7 nM, for PK11195 was 10.9 nM, for diazepam was 191 nM, and for clonazepam was greater than 10(-5) M. Studies in RBL-1 cells also showed one class of saturable binding sites with KD = 1.11 +/- 0.72 nM and Bmax = 177 +/- 69 fmol/10(6) cells; specificity was similar to peritoneal mast cells. Incubation of peritoneal mast cells with Ro5-4864 (10(-10)-10(-5) M) for 60 min, or with 10(-6) M for 0-60 min, showed no change in histamine or serotonin release either in unstimulated mast cells or those stimulated by compound 48/80 or substance P. Experiments with the fluorescent dye Quin-2 showed no change in free intracellular calcium levels in peritoneal mast cells after incubation with Ro5-4864 ranging from 10(-10) to 10(-6) M.