Protein kinase C (PKC) from human leukemia ML-1 cells was found to be susceptible to inhibition by the antineoplastic anthracycline adriamycin (ADR). Half-maximal inhibition (IC50 value) was observed at 200 microM. However, preincubation of ADR with phosphatidylserine (PS) or PKC enzyme, prior to the enzyme assay, reduced the IC50 value from 200 microM to 52 microM or 40 microM, respectively, indicating an affinity of ADR for PS, and also a possible action site for ADR on PKC molecules. Preincubation of ADR with diacylglycerol (DAG) before the PKC assay resulted in a more pronounced effect, i.e., a more rapid decline of PKC activity with an IC50 value of 7 microM. However, the IC50 for ADR inhibition was not altered when ATP, histone or Ca++ were preincubated with ADR. Studies of the kinetic nature of the inhibition revealed that ADR inhibition assumes competitive kinetics with respect to DAG. Therefore, the mechanism by which ADR inhibits PKC activity may involve a multi-site action: a primary interaction with DAG, and a secondary lower interaction with membrane PS and PKC apoenzyme.