Reaction conditions were optimized for the determination of serum glutathione reductase, which has not yet been investigated systematically. Imidazole was found to be the most suitable buffer material; the highest glutathione reductase activity in serum was always obtained with imidazole/HCl buffer, which, in contrast to all other tested buffers, also resulted in the maximal enzyme activity without preincubation. In imidazole buffer, the pH-activity curve of serum glutathione reductase shows a broad optimum between pH 6.5 and 6.9. A GSSG concentration of 2 mmol/l and a NADPH concentration of 0.43 mmol/l gave maximal enzyme activity and a linear reaction over 10 min up to 20 U/l test solution. An investigation of serum glutathione reductase activity from 100 clinically healthy probands gave values between 20 and 50 U/l. In the optimized assay system the glutathione reductase in the serum reacts specifically with GSSG and NADPH.