Incubation of cultured bovine lens epithelial cells (BLECs) in minimal essential medium (MEM) containing 40 mM galactose for 20 hr results in an attenuation of3H-myo-inositol (3H-MI) concentrating ability. Decreased MI uptake could negatively impact on normal phospho-inositide turnover and diacylglycerol production, and presumably, protein kinase C (PKC) activation. The present report examines the relationship between PKC activity, myo-inositol transport and hyperglycemic conditions. PKC activities in the cytosol and particulate fractions of bovine lens epithelial cells in culture were quantitated using a mixed micelle assay following DEAE-cellulose (DE52) and Sephadex G-25 chromatography. Protein kinase C activity was assessed as Ca2+and phospholipid-dependent Ac-myelin basic protein substrate peptide phosphorylation and confirmed using a PKC pseudosubstrate inhibitor peptide (PKC 19–36). Total PKC activity was similar in galactose-incubated cells (871±64 pmol/mg total protein/min) and control cells (881±8 pmol/mg total protein/min) after 20 hr. In unstimulated cells, approximately 90% of the total cellular PKC activity was recovered in the cytosolic fraction. Enzyme translocation was induced with the tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), resulting in a 6-fold increase in membrane-associated PKC activity. A similar PMA-induced translocation was observed in BLECs incubated with 40 mM galactose MEM-maintained cells briefly treated with PMA or the non-phobol PKC activators, SC-10 and mezerein, displayed a rate of3H-MI uptake similar to the untreated control cells. Treatment of galactose-incubated cells with any of the three PKC activators failed to alter the galactose-attenuated3H-MI accumulation. These observations demonstrated the presence of a Ca2+and phospholipid-dependent PKC activity in BLECs that appeared to function equally well in acute hyperglycemic conditions (20 hr) as in cells maintained in physiological medium. Moreover, these studies indicated that MI transport was not regulated directly by PKC and the galactose-induced attenuation in3H-MI-concentrating capability was not a consequence of a derangement in PKC activity under these incubation conditions.