The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-zeta. In addition to primary monocytes, activation of PKC-zeta in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide epsilon and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-zeta from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase. Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-zeta. Furthermore, activation of PKC-zeta failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-zeta activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-zeta is activated downstream of PI 3-kinase in monocytes in response to LPS.