Conventional(c) PKC activity has been shown to increase with skeletal muscle contraction and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC-activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors Calphostin C, Go6976 and Go6983, in incubated mouse soleus and EDL muscles. All potently inhibited contraction-stimulated glucose uptake by 50-100%, while both Go-compounds, but not Calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMPK and eEF2 phosphorylation was unaffected by the blockers. PKC was estimated to account for ~97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKC KO mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared to wildtype. Furthermore, the effects of Calphostin C and Go6983 on contraction-induced glucose uptake were similar in muscles lacking PKC and in wildtype. It can be concluded that PKC, representing ~97% of conventional PKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either non-specific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.