CD28 functions as a cytotoxicity activation receptor in the natural killer (NK) cell line YT-Indy. To analyze the requirement of p56lck kinase in the function of killer inhibitory receptors, we transfected the p56lck negative YT-Indy cell line with the c143 gene encoding for KIR2DL2. Prevanadate treatment revealed KIR2DL2 phosphorylation in the YT-Indy-c143 cell line,as well as SHP1/SHP2 recruitment. YT-Indy-c143 cells were inhibited in their ability to lyse target cells expressing HLA-Cw3, a ligand for KIR2DL2. This inhibition was blocked by anti-KIR2DL2 or anti-HLA class I mAb. CD28 crosslinking on YT-Indy-c143 cells enhanced tyrosine phosphorylation of PLC-gamma 1. The simultaneous ligation of KIR2DL2 with mAb resulted in a decrease in CD28-induced tyrosine phosphorylation of PLC-gamma 1 confirming that dephosphorylation of this protein is involved in the KIR2DL2 induced inhibition of CD28-mediated cytotoxicity. As YT-Indy-c143 cells do not express detectable levels of p56lck, these results indicate that this kinase is not required for transmitting the negative signals generated by KIR2DL2 ligation.