An experiment was conducted to determine the effect of acute heat stress exposure on amino acid digestibility in laying hens. A total of 30 commercial laying hens were singly housed in an environmentally controlled facility, fed a standard laying ration, and exposed to a constant thermoneutral temperature (21 C) for 12 d. The hens were then randomly fed one of three diets (10 hens per diet) and exposed to three consecutive temperature periods (8 d each), which consisted of: 1) a constant 21 C temperature, 2) a cycling temperature of 35 C for 12 h and 29 C for 12 h, and 3) a constant 21 C temperature. The three isonitrogenous (18% CP) diets fed were: 1) a corn-soybean meal diet, 2) a corn-soybean meal diet containing 15% meat and bone meal, and 3) a corn-soybean meal diet containing 5% alfalfa meal and 20% wheat bran. Excreta were collected from all hens during the last 4 d of each temperature period and apparent amino acid digestibility was determined. There was a significant diet effect (P < 0.05) on amino acid digestibility. Digestibility of amino acids in Diet 2 (corn-soybean meal/meat and bone meal) was higher (P < 0.05) than in the other two diets. In addition, digestibility of amino acids in Diet 3 (corn-soybean meal/alfalfa meal/wheat bran) was significantly lower (P < 0.05) than in Diets 1 or 2. Heat stress generally had no significant effect on amino acid digestibility except for His and Lys digestibility. Histidine digestibility was higher during the heat stress period than during the initial and recovery thermoneutral periods, whereas Lys digestibility was higher during the heat stress period than during the initial thermoneutral period. These results indicated that acute heat stress (8 d) had no adverse effects on dietary amino acid digestibility in laying hens.