A fluorimetric assay for D-lactate in human blood samples was developed using an endpoint enzymatic assay with D-lactate dehydrogenase from Staphylococcus epidermidis. The intrabatch and interbatch coefficients of variance were 8.7% (n = 4) and 16.6% (n = 4), respectively. The limit of detection in blood was 3.73 nmol/ml. The assay suffers minor interference from S-D-lactoylglutathione, which was also present in the blood samples. The concentration of D-lactate in blood was (mean +/- SE, nmol/ml) normal healthy individuals, 11.0 +/- 1.2 (n = 7); and diabetic patients, 20.0 +/- 1.3 (n = 55) (a significant increase in diabetes mellitus; P < 0.01, Mann-Whitney U test).