U937 human promonocytic leukemia cells express PKC isozymes β₁, β₂, ε and ζ. Indirect immunocytofluorescence using affinity-purified PKC-specific antibodies indicates that each of the endogenous PKC isozymes in U937 cells display a unique compartmentalization within the intact cell. PKC-β₁ is distributed between two identifiable pools: a cytoplasmic pool which redistributes to the plasma membrane upon activation with acute phorbol ester-treatment, and a membrane-bound pool associated with intracellular vesicles containing β₂-integrin adhesion molecules, cd11b and cd11c. The vesicle-associated PKC-β₁ translocates with the secretory granules to the plasma membrane upon agonist-stimulated activation. PKC-β₂ is associated with the microtubule cytoskeleton in resting cells. PKC overlay assays indicate that PKC-β₂ binds to proteins associated with microtubules, and not directly to tubulin. PKC-ε is associated with filamentous structures in resting cells and redistributes to the perinuclear region upon activation with phorbol esters. In differentiated U937 cells, PKC-β₁ remains associated with vesicles translocat-ing from the trans-Golgi region to the plasma membrane and PKC-ε is primarily associated with perinuclear and plasma membranes. PKC-ζ, which does not respond to phorbol ester treatment, is primarily cytosolic in undiffer-entiated cells and accumulates in the nucleus of differenti-ated cells blocked in the G2 phase of the cell cycle. The data clearly demonstrate that individual PKCs localize to different subcellular compartments and promote the hypothesis that PKC subcellular localization is indicative of unique functions for individual PKC isozymes.