Broilers chickens have limited ability to utilize phytate phosphorus (PP), and the influence of nutrients on the activity and efficacy of intestinal phytase is unclear. Therefore in a 2 x 2 x 2 factorial experiment, male chicks were fed 0 or 0.21 mg/kg 25-hydroxycholecalciferol (25-OH D3), 4 or 9 g/kg Ca supplied from CaCO3 or Ca malate from 14 to 24 d of age (six pens/treatment, eight birds/pen). Source of Ca had no effect on tibia ash, intestinal phytase activity, or apparent ileal PP hydrolysis. Phytase activity (Vmax) within brush border vesicles prepared from small intestinal mucosa was greater in chicks fed 4 vs. 9 g/kg Ca (P < or = 0.05). Similarly, birds fed 4 vs. 9 g/kg Ca were able to hydrolyze an additional 24.36% PP (P < 0.01). Intestinal phytase activity and apparent ileal PP hydrolysis were not affected by 25-OH D3 supplementation, but tibia ash was improved by 2.7% (P < 0.01). A subsequent experiment validated the effect of dietary Ca (4 and 9 g/kg) and elucidated differences between strains (Ross 308 and Hubbard x Peterson) from 8 to 22 d of age (six pens/treatment, eight birds/pen). The strains responded similarly to dietary Ca in terms of intestinal phytase and apparent ileal PP hydrolysis. Intestinal phytase activity was 9% greater in birds fed 4 vs. 9 g/kg Ca (P < 0.05) and apparent ileal PP hydrolysis was 11.9% greater (P < 0.02). In conclusion, a typical dietary Ca concentration (9 g/kg) reduced intestinal phytase activity and apparent ileal PP hydrolysis.