1-beta-D-arabinofuranosylcytosine (ara-C) conjugates of phospholipid were shown to be highly antineoplastic against various tumor cells. In this study, we report that these conjugates are potent activators of protein kinase C (PKC, EC) in vitro. Although required Ca2+, PKC activation by the conjugates occurred even in the absence of phospholipid and diacylglycerol. Among the conjugates, 1-beta-D-arabino-furanosylcytosine 5'-diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol [(ara-CDP-DL-PBA); ara-C conjugate of ether phospholipid], was employed to investigate its mode of activation, since ether phospholipid has been reported to be a regulator of the PKC. When PKC was activated by ara-CDP-DL-PBA, diacylglycerol enhanced its activity with 3-fold reduction of an apparent Ka value for ara-CDP-DL-PBA and no change in the Vmax. During the PKC activation by phosphatidylserine, ara-CDP-DL-PBA exhibited a synergistic effect on the activation. Studies on the relationship between the structures of ara-CDP-DL-PBA and their effects on PKC activity showed that phosphate group of ether lipid was important for its activation of PKC, and that conjugation of ara-C and ether lipid further enhanced the enzyme activity. These results suggest that the ara-C conjugate of phospholipid activates PKC in a co-operative manner with diacylglycerol and/or phosphatidylserine, however, the exact mechanism of the antineoplastic effect of ara-CDP-DL-PBA through PKC activation still remains speculative.