The effect of thiamine deficiency on glyoxylic acid metabolism in mice and rats was investigated to determine whether the vitamin deficiency results in gross effects on glyoxylate levels via an alteration in the activity of α-ketoglutarate:glyoxylate carboligase. Thia-mine-deprived or pyrithiamine-treated mice did not show a decreased oxidation of [1-¹⁴C]glyoxylate to respiratory CO₂; there was some decrease in the conversion of [2-¹⁴C]glyoxylate into CO₂ by pyrithiamine-treated mice, but not by thiamine-deprived animals. Dietary thiamine deprivation caused a decrease in carboligase levels in liver but no effect on levels in three brain regions. Pyrithiamine treatment had no significant effect on liver carboligase activities, but did decrease the levels in cerebrum, cerebellum and brainstem. Thiamine-deprived and pyrithiamine-treated mice showed decreased urinary glycolic acid excretion. Glyoxylic acid excretion by thiamine-deprived rats was monitored in order to re-examine a previous report by another laboratory that glyoxyluria occurs under these conditions. Trace amounts of glyoxylate could be detected in the urine of rats fed thiamine-deficient diet for 3–5 weeks, but urinary glyoxylate was not detectable at later stages of thiamine deprivation. These results do not support a significant role for α-ketoglutarate:carboligase activity in the primary etiology of thiamine deficiency syndromes.