Glyoxalase I (EC 184.108.40.206) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. The KM value for methylglyoxal-glutathione hemithioacetal was 192 +/- 8 microM and the kcat value was 10.9 +/- 0.2 x 10(4) min-1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had a Ki value of 0.16 +/- 0.04 microM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with a Ki value of 3.12 +/- 0.88 microM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with a Ki value of 18 +/- 8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.