Zucker diabetic fatty (ZDF) rats develop non-insulin-dependent diabetes mellitus concomitantly with loss of glucose responsiveness and GLUT-2, the high-Michaelis constant glucose transporter of beta-cells. To determine the integrity of beta-cell glucose metabolism distal to the level of glucose transport and phosphorylation, we examined the insulin responses of isolated pancreata to 5, 10, and 20 mM D-glyceraldehyde and monomethylsuccinate, as well as to glucose. The insulin response of diabetic pancreata to glucose was 90% below the response prior to the onset of diabetes, whereas the responses to glyceraldehyde and succinate had declined to 65 and 44%, respectively, below the prediabetic responses. D-[14C]glyceraldehyde oxidation by diabetic islets was 74% below that of islets from lean nondiabetic controls. We conclude that 1) the insulin responses to glyceraldehyde and monomethylsuccinate, as well as to glucose, are impaired in the diabetes of ZDF rats and 2) the impairment of the glucose response was greater than that of the glyceraldehyde response, which was, in turn, greater than that of the monomethylsuccinate response; this decrescendo pattern of impairment is consistent with defects at multiple sites in glucose metabolism; if the defect were entirely due to a postmetabolic signaling defect, the impairment to glucose and its metabolites should be comparable.