Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent K(m) values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.