Insulin and 12-O-tetradecanoyl phorbol-13-acetate (TPA) induce both glucose uptake and translocation of protein kinase C (PKC) from cytosol to membrane in insulin-sensitive tissues as previously reported by several investigators. We examined insulin-mediated PKCβI, βII, and ε translocation from cytosol to cytoskeleton, and expression of PKC α, βI, βII, γ, and ε isoforms using the reverse transcription polymerase chain reaction (RT-PCR) method during treatment with insulin for 240 min in rat adipocytes. Insulin-induced increases in PKCβI, βII, and ε were greater in the cytoskeleton fraction than those in the membrane fraction. Insulin induced time-dependent increases in PKCα, γ, ε and ζ mRNA levels for up to 240 min (555%, 117%, 236% and 138% increase, respectively). TPA also induced time-dependent increases in PKC α and γ (34% and 500% increase, respectively) but not in PKCζ. However, PKCβI mRNA was decreased for up to 60 min and then maintained at under the basal level during stimulation with insulin and TPA. On the other hand, PKCβII mRNA was markedly increased for up to 240 min. These results suggest that insulin-regulated PKC α, γ and ε mRNA levels and PKCβ mRNA alternative splicing may occur in rat adipocytes.