The b-isoform of protein kinase C (PKC) has paradoxically been suggested to be important for both insulin action and insulin resis- tance as well as for contributing to the pathogenesis of diabetic com- plications. Presently, we evaluated the effects of knockout of the PKCb gene on overall glucose homeostasis and insulin regulation of glucose transport. To evaluate subtle differences in glucose homeosta- sis in vivo, knockout mice were extensively backcrossed in C57BL/6 mice to diminish genetic differences other than the absence of the PKCb gene. PKCb2/2 knockout offspring obtained through this back- crossing had 10% lower blood glucose levels than those observed in PKCb1/1 wild-type offspring in both the fasting state and 30 min after ip injection of glucose despite having similar or slightly lower serum insulin levels. Also, compared with commercially obtained C57BL/ 6 -129/SV hybrid control mice, serum glucose levels were similar, and serum insulin levels were similar or slightly lower, in C57BL/6 - 129/SV hybrid PKCb knockout mice in fasting and fed states and after ip glucose administration. In keeping with a tendency for slightly lower serum glucose and/or insulin levels in PKCb knockout mice, insulin-stimulated 2-deoxyglucose (2-DOG) uptake was enhanced by 50 -100% in isolated adipocytes; basal and insulin-stimulated epitope-tagged GLUT4 translocations in adipocytes were increased by 41% and 27%, respectively; and basal 2-DOG uptake was mildly increased by 20 -25% in soleus muscles incubated in vitro. The reason for increased 2-DOG uptake and/or GLUT4 translocation in these tissues was uncertain, as there were no significant alterations in phosphatidylinositol 3-kinase activity or activation or in levels of GLUT1 or GLUT4 glucose transporters or other PKC isoforms. On the other hand, increases in 2-DOG uptake may have been partly caused by the loss of PKCb1, rather than PKCb2, as transient expression of PKCb1 selectively inhibited insulin-stimulated translocation of epitope-tagged GLUT4 in adipocytes prepared from PKCb knockout mice. Our findings suggest that 1) PKCb is not required for insulin- stimulated glucose transport; 2) overall glucose homeostasis in vivo is mildly enhanced by knockout of the PKCb gene; 3) glucose transport is increased in some tissues in PKCb knockout mice; and 4) increased glucose transport may be partly due to loss of PKCb1, which nega- tively modulates insulin-stimulated GLUT4 translocation. (Endocri- nology 140: 4470 - 4477, 1999)