Although nine diacylglycerol kinase (DGK) isozymes have been identified, our knowledge of their individual functions is still limited. Here we report that the levels of DGKgamma mRNA/protein in human leukemia HL-60 and U937 cells were rapidly and markedly decreased upon cellular differentiation into macrophages. In contrast, the enzyme expression remained almost unchanged in granulocytic differentiation pathway. Interestingly, the overexpression of wild-type or constitutively active DGKgamma, but not its kinase-dead mutant, markedly inhibited phorbol ester-induced cell attachment and nonspecific esterase activity, which are hallmarks of macrophage differentiation. We noted in this case that no effects were observed for the corresponding constructs of a closely related isozyme, DGKalpha. Prior to the cell attachment, phorbol ester induced translocation of DGKgamma from the cytoplasm to the cell periphery, resulting in its co-localization with F-actin together with protein kinase Cdelta. The results suggest that DGKgamma negatively regulates macrophage differentiation through its catalytic action operating on the cytoskeleton.