Glomerular mesangial cells (MCs) have been used as an in vitro model for glomerular disease. The culture conditions used for these cells vary and include the use of insulin or insulin-transferrin-selenous acid (ITS) in the growth medium. We studied the effect of ITS in the growth medium containing either normal or high glucose on the expression of protein kinase C (PKC) isoforms in MCs. In the presence of ITS in the medium, MCs expressed lower levels of both PKC isoforms in their cytosol in comparison to MCs grown in medium without ITS. Upon stimulation with PMA, both isoforms were translocated to the particulate (nucleus/cytoskeleton) compartment in MCs grown in presence of ITS. However, in the absence of ITS in the growth medium, both PKC isoforms were primarily translocated to the membrane compartment upon PMA stimulation. These results indicate that insulin in the growth medium may activate MCs resulting in translocation of PKC from the cytosol to other subcellular compartments. This effect is even evident in MCs grown in normal glucose concentration. Our data indicate that the use of ITS in growth medium and eventual interpretation from such experiments involving primary mesangial cells grown in culture needs careful evaluation.