Protein kinase C (PKC) epsilon in 3T3 and 3T6 fibroblasts and in C6 glioma cells migrated on SDS/PAGE predominantly as a doublet with molecular masses of 87 and 95 kDa (PKC epsilon(87) and PKC epsilon(95) respectively). PKC epsilon(95) predominates when cells reach confluency but PKC epsilon(87) was the main form detected within 15 min when confluent cells were passaged at low cell density into fresh medium containing serum and allowed to adhere. Matrix-assisted laser-desorption ionization-time-of-flight MS analysis and experiments with phosphospecific antibodies revealed that PKC epsilon(87) is phosphorylated at Thr-566 and Ser-703, and PKC epsilon(95) is additionally phosphorylated at Ser-729. Cell fractionation studies revealed that PKC epsilon(95) is associated with the nuclear fraction, whereas PKC epsilon(87) was found in the 100,000 g cytosol fraction. Immunofluorescence studies confirmed these findings and showed that PKC epsilon(95) had a perinuclear, probably Golgi, localization and PKC epsilon(87) was distributed in the cytosol. It is proposed that phosphorylation at Ser-729 may be important for determining the intracellular localization of PKC epsilon, and that a specific Ser-729 phosphatase may be activated on cell passage to convert PKC epsilon(95) to PKC epsilon(87).