Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibit spontaneous cytotoxicity mediated by human natural killer (NK) cells. The aim of this study was to determine which stage(s) of the NK cytotoxic reaction is inhibited by these compounds. Effector/target cell binding studies performed in parallel with cytotoxicity assays using purified large granular lymphocytes indicated that inhibition is a post-binding event. The kinetic profile of inhibition in a calcium pulse assay showed that inhibition continues long after the effector cell triggering stage and that although sulfasalazine may have some inhibitory effect on the calcium-dependent events of the programming phase, sulfapyridine continues to inhibit during the calcium-independent or lethal hit phase of the cytotoxic sequence. The NK soluble cytotoxic factor (NKCF) assay was used as a measure of the lethal hit since the time course of this assay permits study of the various substages of this terminal event in the lytic sequence. Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibited NKCF-mediated target cell lysis. Different substages of the NKCF-induced lytic reaction were affected by these agents. Sulfasalazine appears to inhibit binding of NKCF to the target cell whereas sulfapyridine predominantly inhibits early post-binding events.