Three experiments were conducted to determine the possible interaction of particle size, in vitro limestone solubility rate, and the Ca status of the hen, respectively, with dietary Ca level and in vivo Ca solubilization in laying hens. The in vivo Ca solubilization in hens was determined by subtracting Ca recovered as limestone in the excreta (by repeated washing) from Ca fed as limestone. Limestones of two different particle sizes [.5 to .8 mm (small) versus 2 to 5 mm (large)] with the same in vitro solubility rates were used in Experiment 1. Limestones of two different in vitro solubility rates (4 versus 10%/15 min in .079 N HCl) with the same particle size in Experiment 2 were assessed for in vivo Ca solubilization. In both experiments, laying hens were fed diets containing graded levels of Ca: 1.5, 3.0, 4.5, and 6.0%. In Experiment 3, hens fed a Ca-deficient (1%) diet for 1 wk were compared with hens fed a diet containing 3.75% Ca to assess in vivo Ca solubilization as influenced by graded dietary Ca levels: 1.5, 3.0, and 4.5% Ca. In vivo Ca solubilization in hens was influenced (P < .05) by dietary Ca level (inverse relationship), limestone particle size (large particle greater than small particle), and Ca status of hens (Ca-deficient hens greater than normal hens). In vivo Ca solubilization in laying hens, for the particle size tested, was not influenced by in vitro limestone solubility rate. Dietary Ca level had a significant interaction (P < .05) with limestone particle size (Experiment 1) and with Ca status of hens (Experiment 3) in influencing in vivo Ca solubilization in laying hens.