Ketone bodies (KBs) serve as alternative substrates when glucose availability is compromised (e.g. starvation). It is unclear whether KBs directly contribute to insulin resistance. We investigated the effects of D,L- beta-hydroxybutyrate (BOH, the major KB in vivo) on glucose transport in isolated mouse soleus (oxidative) and extensor digitorum longus (EDL, glycolytic) muscle. BOH did not alter glucose transport (2-deoxyglucose uptake) in a noteworthy fashion in EDL muscle under any condition studied, but inhibited insulin- mediated transport in soleus in a time and concentration-dependent manner. Under optimal conditions (19.5 h exposure to 5 mM BOH), insulin (20 mU/ml)-mediated glucose transport was almost fully inhibited (control=6.10.4 mmol/ml intracellular water/30 min; BOH=2.80.2; P<0.001). The inhibitory effect was reproduced with D- but not with L-BOH. BOH did not significantly affect hypoxia- or AICAR (activates AMP-dependent protein kinase)- mediated glucose transport. The BOH effect did not require the presence or utilization of glucose since it was also seen when glucose in the medium was substituted with pyruvate. The BOH effect was not reversed by an exogenous antioxidant and was not associated with increased production of reactive oxygen/nitrogen species. BOH did not alter the levels of total tissue GLUT-4 protein, but blocked the insulin- mediated phosphorylation of PKB by almost 50 %. This inhibition was not associated with the phosphorylation of PKC delta. These data demonstrate that BOH inhibits insulin-mediated glucose transport in oxidative muscle by inhibiting insulin signaling. Thus KBs may be potent diabetogenic agents in vivo.