Protein Kinase C. Using protein kinase C-depleted cytosol, PMA-dependent activation was lost, and added protein kinase C reconstituted PMA-dependent phospholipase D activity. Unlike phospholipase D in some other tissues which show ATP-independent activation by protein kinase C, activation using neutrophil subcellular fractions required ATP and was inhibited by staurosporine, implying that protein kinase C is functioning by classical phosphorylation-dependent mechanisms. The phosphorylation target was shown to be in the plasma membrane, and may be the phospholipase D itself. Calcium-dependent PKC isoforms, particularly the beta isoform, reconstituted activity, whereas calcium-independent and atypical isoforms were not capable of reconstituting activity.