Activation of protein kinase C (PKC) by bacterial lipopolysaccharide had recently been implicated in the pathogenetic sequence of gram-negative sepsis, endotoxicosis, hyperinsulinism, and the alterations in glucoregulation that eventuate in glucose dyshomeostasis. This study used the peptide antibiotic polymyxin B (PMX-B) and H-7, an isoquinoline sulfonamide, as inhibitors of PKC activation to evaluate responses to provocative insulin and glucose tolerance tests in control vs. endotoxic rats. Fed male rats were treated with either Salmonella enteritidis endotoxin (ETX; 0.33 mg/kg iv) or saline 120 min before intravenous insulin tolerance testing (IVITT) with human insulin (1 U/kg) or intravenous glucose tolerance testing (IVGTT) with D-glucose (1.2 g/kg). H-7 in dimethyl sulfoxide at 25 mg/kg, PMX-B in saline at 0.25 mg/kg, or the respective vehicles were administered 5 min before the tolerance tests. Neither H-7 nor PMX-B had any significant acute effects on basal plasma glucose or lactate values. The decline in plasma with IVITT was augmented by ETX; however, concomitant H-7 or PMX-B attenuated the insulin hypoglycemia. The computed half-life of glucose in the IVGTT was decreased by ETX; however, concomitant H-7 or PMX-B decreased the tolerance alteration. In addition, both H-7 and PMX-B attenuated the rise in insulin induced by the IVGTT. Thus the hyperinsulinism and the glucoregulatory disturbances in endotoxicosis may be mediated by PKC activation and ameliorated by PKC inhibition.