Alternative splicing of pre-mRNA encoding the carboxy-terminal (C-terminal) exons of protein kinase C beta (PKC beta) leads to the expression of two protein isoforms, PKC beta 1 and PKC beta II, with the potential for different functions. PKC beta II expression is regulated by insulin via alternative mRNA splicing. A physiological consequence of its activation was investigated in L6 rat skeletal muscle cells expressing GLUT4 transporters, a cell line in which PKC is involved in glucose transport. We examined the contribution of PKC beta II for insulin-stimulated [3H]2-deoxyglucose uptake by constructing three PKC beta II C-terminal deletion mutants designated M216, M217, and M218. When transiently expressed in COS1 cells, M217, with nine amino acids deleted, demonstrated autophosphorylation activity 10-fold less than full-length PKC beta II. The mutants M218, with 13 amino acids deleted, and M216, with 52 amino acids deleted, demonstrated no autophosphorylation activity and are kinase negative. When transiently expressed in L6 myotubes, M217 inhibited insulin-stimulated 2-deoxyglucose uptake by 45% (with a 45% transfection efficiency) whereas M216 and M218, kinase-negative mutants, had no effect compared with cells transfected with control plasmid. Cotransfection of full-length PKC beta II with M217 was able to rescue the inhibition of insulin-stimulated 2-deoxyglucose uptake as compared with cotransfection of M217 with the control plasmid, suggesting that M217 acts as a dominant-negative. In contrast, cotransfection of full-length PKC beta I, the other alternatively spliced form, did not rescue inhibition of insulin-stimulated 2-deoxyglucose uptake by M217. To further demonstrate the involvement of PKC, specifically PKC beta II, in insulin-stimulated 2-deoxyglucose uptake, we used two inhibitors, CG41251 (a specific PKC inhibitor) and CG53353 (a PKC beta II-specific inhibitor at 1 microM). Both inhibited insulin-stimulated 2-deoxyglucose uptake 50-60% in L6 myotubes. We conclude that M217 may act as a specific PKC beta II dominant-negative and that PKC beta II is more specific for insulin-stimulated 2-deoxyglucose uptake in these cells than PKC beta I.