A study was conducted to determine the in vivo and in vitro limestone solubility of different mixtures of small and large particle limestone. Small (0 – 1.0 mm) and large (2.0 – 3.8 mm) particles were blended to obtain five distribution mixtures consisting of 0%, 25%, 50%, 75% and 100% large particles limestone. Thirty three individually caged Lohmann-Silver laying hens were randomly allocated to each of the five treatments for the determination of in vivo limestone solubility. A hydrochloric acid (HCl) solution was used as solvent for in vitro limestone solubility determination. An increase in the percentage large particle limestone resulted in a significant increased in vivo and decreased in vitro limestone solubility. A significant increased intestinal limestone content (ILC) and decreased faecal limestone content (FLC) were observed with an increase in the percentage large particle limestone. These results suggest that an increased percentage of larger particle limestone in distribution mixtures, resulting in a prolonged retention time in the gizzard, could provide more Ca2+ to laying hens due to the increased in vivo limestone solubility rate thereof.