Abstract: Three in vitro experiments were carried out to determine the effect of temperature, moisture content, duration of incubation, Ca level, soaking with water or citric acid onphytate degradation in a wheat–barley–rye–soybean meal-based broiler diet. In experiment 1, phytase activity of individual feed ingredients and 4 low-P broiler diets, containing 2, 4, 8, and 12g Ca per kg diet, respectively, were measured in the presence or absence of sodium phytate or soybean meal. By using sodium phytate as substrate, phytase activity of rye, wheat, barley and soybean meal was 3350, 1170, 580 and 30FTU/kg, respectively (P<0.001). Calcium level had no effect on the activity of intrinsic phytase of diets (P>0.05). In experiment 2, the effect of 2 moisture levels (0.25 and 50%), 3 temperatures (70, 75 and 80̊C) and 3 durations of incubation (2, 4, and 8min) on the residual phytase activity of diet 1 (basal diet) were evaluated as a 2×3×3 factorial arrangement with 3 replicates per treatment. The loss of activity of intrinsic phytase increased from 0.25 at 70̊C to 0.61 at 80̊C (P<0.001). Increasing duration of incubation from 2min to 8min increased the loss of activity from 0.27 to 0.52 (P<0.001). By increasing the moisture content, loss of activity of intrinsic phytase also significantly increased from 0.25 at 25% moisture to 0.53 at 50% moisture. In experiment 3, the effects of 4 dietary Ca levels (2, 4, 8, and 12g/kg), 2 types of soaking (with water or citric acid solution) and 4 duration times of soaking (2, 4, 8 and 24h) on the amount of P released from the complete diet were determined as a 4×2×4 factorial arrangement with 3 replicates per treatment. Increasing Ca level of the diet from 2 to 12g/kg decreased the amount of released P from 1.19 to 0.97g/kg of diet (P<0.001). As time of soaking increased, the difference in released P due to soaking with citric acid comparison to soaking with deionized water became more prominent (interaction soaking×time; P<0.05). In conclusion, soaking of a broiler diet, especially in a citric acid solution one day before feeding, may increase available P and decrease the need of supplemental inorganic P to these diets, thus improving the sustainable use of P resources. [Copyright &y& Elsevier]