The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.