Three groups of 10 age- and sex-matched nondiabetic volunteers took 0, 750, or 1500 mg of vitamin C each day for 12 weeks. Glycohemoglobin (GHb) was measured by HPLC, electrophoresis, affinity chromatography, and immunoassay at baseline (-4 weeks and -1 day), during supplementation (6 weeks and 12 weeks), and after supplementation ended (6 and 12 weeks). Plasma vitamin C increased twofold during supplementation but, in contrast with the results of Davie et al. (Diabetes 1992; 41:167-73), there were no between-group differences in GHb, glucose, and fructosamine concentrations. Fructosamine may have increased with storage time. The net effects of vitamin C on absolute GHb at 12 weeks vs -1 day (and at 12 weeks vs 12 weeks after) in % GHb amounted to: HPLC -0.035 (-0.050); electrophoresis +0.005 (+0.035); affinity chromatography -0.070 (+0.015); and immunoassay -0.110 (+0.025). We conclude that supplementation of nondiabetics with 750 or 1500 mg of vitamin C daily for 12 weeks does not cause interference in GHb determinations by HPLC, electrophoresis, affinity chromatography, or immunoassay, and does not reduce in vivo Hb glycation.