The subchronic oral toxicity of glyoxal via drinking water and the effect on in vivo protein synthesis in tissues following a single treatment with this substance were assessed in Sprague-Dawley male rats. Animals received drinking water containing glyoxal levels of 2000, 4000, and 6000 mg/liter ad libitum for 30, 60, and 90 days in Phase I. In Phase II, the high-dose and control-1 groups fed the diet ad libitum, and a diet-limited control-2 group given the same amount of diet as consumed by the high-dose group were maintained for 90 and 180 days. The study designs included observations of clinical signs, body weights, major organ weights, gross and histopathological examinations, serum clinical chemistry, and biochemical examinations such as glyoxalase activity and glutathione content in selected tissues. Body weight gain and organ weights significantly decreased with dosage. Although consumption of food and water was also depressed in the exposed group, the reduction of body weight gain was greater in the high-dose group than in the diet-limited control 2 group. Histopathological examinations revealed only a slight papillary change in the kidneys from the high-dose group at both 90 and 180 days terminations in Phase II. The induction of both glyoxalase I and II was observed in liver and erythrocytes at 30-day termination of the exposed groups. Serum enzyme and protein levels were significantly reduced by the mid- and/or high-dose exposures. With a single oral high-dose treatment of glyoxal, a great decline in the incorporation of L-[3H]leucine was shown particularly in the liver, and this probably led in part to a reduction in the serum protein levels in rats following subchronic exposure to glyoxal. These data indicated an overall low degree of systemic toxicity to rats exposed subchronically to glyoxal via drinking water.