This study was undertaken to investigate whether target cell class I HLA antigen expression induced by phorbol ester and interferon-α (IFN-α) was associated with resistance to natural killer (NK) cells and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Class I antigen expression on the surface of the K562 erythroleukemia cell line was enhanced by either IFN-α or phorbol ester (PDBu). Addition of PDBu together with IFN-α had a synergistic effect on class I antigen expression on the cells. Furthermore, synergism between IFN-α and PDBu was also found in class I antigen expression by MOLT-3 cells. This synergistic effect on class I antigen expression was blocked by the protein synthesis inhibitor (cycloheximide). Pretreatment of K562 cells with PDBu and IFN-a made them more resistant to lysis by NK and LAK cells than did either PDBu or IFN-α. In contrast to PDBu, 4αPDD, a biologically inactive phorbol analogue, alone or combination with IFN-α, had no effect on class I antigen expression and susceptibility to lysis by NK and LAK cells. Kinetic experiments showed an inverse relationship between the expression of class 1 antigens and susceptibility to NK cell-mediated cytolysis. Using cold target competition analysis, target cells pretreated with PDBu and IFN-α clearly competed less effectively than did untreated cells for lysis of untreated target cells. These results demonstrate that target cells pretreated with PDBu and IFN-a decrease their sensitivity to natural killer and lymphokine-activated killer cells inversely with target cell class I HLA antigen expression.