The pharmacokinetic values of d,l-leucovorin and l-leucovorin were compared in eight healthy volunteers following oral administration of 25 mg d,l-leucovorin and 12.5 mg l-leucovorin. Serum levels of l-5-formyltetrahydrofolate, l-5-methyltetrahydrofolate, and total reduced folates were measured by an established microbiological method. Pharmacokinetic data for both preparations were consistent with those previously reported for d,l-leucovorin, with essentially complete first pass metabolism to l-5-methyltetrahydrofolate, the active metabolite. No differences were found between the two preparations in serum concentrations of active folate fractions, AUC, or Cmax, or in clearance and volume of distribution estimates. These data suggest that after administration of 25 mg of d,l-leucovorin, the d-diastereoisomer has no significant effect on the standard pharmacokinetic measurements of the active l-folates.