Natural killer (NK) cell-resistant tumors exist despite their ability to bind cells from the effector population. Tumor sensitivity to NK activity was therefore examined at the level of susceptibility to cytolysin-containing NK cell cytotoxic granule extracts. The NK-sensitive SL2-5 murine lymphoma was markedly more susceptible than the NK-resistant L5178Y-F9 to solubilized granule preparations from the rat NK tumor cell line RNK-16, and this corresponded also with tumor sensitivity to hypotonic lysis. However, the resistant L5178Y-F9 was better able to inhibit the extract activity than the SL2-5. Dissociation of the binding and lysis phases of the cytolysin reaction based on their differential temperature requirements, 4 degrees C for binding and 37 degrees C for lysis, permitted an examination of the cytolysin/tumor interaction prior to lysis. The residual cytotoxic activity was lower after extract exposure to the L5178Y-F9 compared with the SL2-5 consistent with possible inhibitor production. Finally, supernatant material collected from the L5178Y-F9 was a better inhibitor of granule extract lysis and acted preferentially in the extract-binding phase. The inhibitor appears to be protein in nature, relatively stable, and exhibits molecular weight heterogeneity ranging from 2000 to greater than 300,000.