The antineoplastic agent, hexadecylphosphocholine, a phospholipid analogue, inhibited phosphatidylserine-activated protein kinase C in vitro at concentrations higher than 40 mumol/l. The half-inhibitory concentration (IC50) was 62 mumol/l. Another alkylphosphocholine, dodecylphosphocholine, did not have an inhibitory effect on protein kinase C. At the same concentrations, hexadecylphosphocholine antagonised the phorbol ester-stimulated proliferation of Madin-Darby canine kidney cells whereas dodecylphosphocholine had no effect. In addition, phorbol ester-induced morphological changes of these epithelial cells were antagonised by hexadecylphosphocholine. Both effects of hexadecylphosphocholine, the inhibition of protein kinase C and the antagonisation of the altered cell morphology induced by phorbol ester, were comparable to those observed after treatment with sphingosine, a known protein kinase C inhibitor. We conclude that one possible mechanism of the antineoplastic action of hexadecylphosphocholine is mediated by inhibition of protein kinase C.