The effects of ruthenium red (RR) on inositol 1,4,5-trisphosphate (InsP 3)-induced responses were studied in rat bone marrow megakaryocytes with the patch-clamp whole-cell recording technique in combination with fura-2 microfluorometry. Internal application of InsP 3 (100 μM) increased intracellular Ca 2+ concentration ([Ca 2+] i) and activated the Ca 2+-dependent K + current. Administering InsP 3 together with RR (100–500 μM) inhibited InsP 3-induced responses (both Ca 2+ and current responses) in a dose-dependent fashion. Pretreatment of megakaryocytes with extracellular RR (50 μM) also inhibited InsP 3-induced responses. Intracellular and extracellular application of RR reduced ADP-induced increases in [Ca 2+] i. In contrast, in isolated single pancreatic acinar cells, RR had no effect on InsP 3-induced responses. Taken together, these results suggest that the site of the inhibitory action of RR is at the InsP 3 receptor, or its closely associated proteins. In addition, we have shown that RR is a useful pharmacological tool with which to examine the InsP 3-mediated responses of megakaryocytes.