Microbial phytase is a prominent feed enzyme used in animal feeds, but there is relatively little information on its use in laying hen diets. In this experiment, an Escherichia coli6-phytase (Quantum) was evaluated for its efficacy in a 40-wk laying hen production trial. A total of 1,080 White Leghorn hens (540 each of Shaver and Bovan strains) were fed mash corn-soybean meal-based diets containing 0.35% (positive control, PC), 0.25% (negative control, NC1), or 0.15% (NC2) nonphytate phosphorus (NPP). Six more diets were manufactured by supplementing the negative control diets with 200, 400, and 600 U/kg of exogenous phytase, resulting in a total of 9 treatments. Each dietary treatment × strain subclass was replicated 4 times with 5 adjoining cages per replicate (3 hens per cage) in a randomized complete block design. Production performance was measured from 21 to 61 wk of age. Only minor differences in production characteristics were found between the PC and NC1 treatments regardless of phytase addition, indicating that 0.25% NPP resulted in P intake that was at or above the hen's requirement. In contrast, the hens fed 0.15% NPP diet without phytase supplementation had significantly (P< 0.05) reduced total hen housed egg production and body weight at 61 wk of age in comparison to the PC treatment, whereas the incidence of soft-shelled, cracked, and broken eggs was increased significantly (P< 0.05) in hens fed the NC2 diet. Addition of phytase to the NC2 diet improved these production characteristics to levels equal or better than the PC diet. The results indicated that Quantum phytase was efficacious in corn-soybean meal-based diets fed to White Leghorn laying hens and can be used to reduce diet supplementation with inorganic phosphorus.