The effects of different pelleting temperatures on the activity of cellulase, bacterial amylase, fungal amylase, and pentosanase were tested. Samples of a commercial barley-wheat-soybean diet containing different enzyme preparations were pelleted at 60, 70, 80, 90, and 100 C (pellet temperature measured at the die outlet) through a die containing holes 2.5 mm in diameter. Enzymatic analyses were conducted on either soluble substrates or by measuring the ability of the tested enzymes to decrease the viscosity of the diet. Measurements made on soluble substrates suggest that cellulase, fungal amylase, and pentosanase maintained activity when being pelleted at temperatures up to 80 C and bacterial amylase maintained activity at temperatures up to 90 C. Pentosanase and amylases showed little or no effect on the viscosity of the diet. Cellulase addition decreased the viscosity at all temperature levels, even after being pelleted at 90 and 100 C (P < 0.05). No cellulolytic activity was detected on the soluble substrate after these pelleting temperatures. Measurements on a soluble substrate might therefore not always reflect the true stability of a preparation because the ability of a carbohydrase to decrease the viscosity of the digesta is important to its effect in the gastrointestinal tract. Measurements on soluble substrates suggest that cellulase, fungal amylase, and pentosanase can be pelleted at temperatures up to at least 80 C and bacterial amylase up to 90 C without a considerable loss in analyzed activit