We hypothesized that serum D-lactate may be increased in vivo in diabetes mellitus as a result of increased glucose flux through the glyoxalase pathway and/or via hepatic ketone metabolism. Levels of D-lactate and related metabolic intermediates were measured in 30 cats with spontaneous diabetes mellitus and in one ketoacidotic nondiabetic cat. Serum D-lactate was significantly (P = .0051) elevated in cats with ketoacidosis (337.2 +/- 70.2 mumol/L) as compared with nonketoacidotic diabetic (140.3 +/- 58.8) and control (25.0 + 6.5) cats. Two nonketoacidotic cats also had high levels of D-lactate. There was a significant linear correlation (r = .684, P = .0001) between D-lactate and beta-hydroxybutyrate concentrations. Serum D-lactate did not correlate with serum glucose (r = .078, P = .6825), and in vitro erythrocyte D-lactate formation did not increase in the presence of hyperglycemia. These data suggest that hepatic ketone metabolism, rather than hyperglycemia, may be a major source of serum D-lactate in diabetics.