Major histocompatibility complex (MHC) class I antigens on tumour cell surfaces have been shown to modulate target susceptibility to natural killer (NK) cell-mediated lysis in some, although not all, systems investigated. MHC class II expression may also affect NK cell function, but the mechanism by which MHC class II antigen regulates NK cell activity has not been fully examined. In this study we induced HLA-DR1 expression by gene transfection into the classic NK-sensitive K562 cell line to study the interaction of NK cells with MHC class II molecules and the effect of brefeldin-A (BFA), an endogenous antigen-processing pathway blocker, on NK-target cell interaction. We demonstrated that the expression of HLA-DR1 on the cell surface reduced K562 cell susceptibility to NK lysis by peripheral blood monuclear cells and a NK cell line. The effect was demonstrable in prolonged (8 hr) cytotoxicity assays and was blocked by pretreatment of target cells with anti-HLA-DR antibody. Treatment of K562 DR transfectant with BFA abrogated the resistance of K562 transfectant to NK-mediated cytolysis. These findings indicate that HLA class II molecules regulate NK cell function and target recognition, and suggest that endogenous peptides presented through MHC molecules are responsible for regulating NK cytolysis.