Previous reports suggest that the site of the energy-dependent intestinal calcium transport against an electropotential and concentration gradient is located along the basal-lateral membrane of the mucosal cell. Accordingly, basal-lateral membranes were prepared from rat intestinal homogenates in order to identify the enzyme mediating this step in the transport process. An alkaline phosphatase was delineated which utilized ATP as a substrate and was dependent on both Na^- and Ca⁺⁺ with optimum enzyme activity at 200 mM and 0.04 mM, respectively. Furthermore, the activity of the enzyme was demonstrated to decrease with the advance in age of the animal and to decrease with removal of the parathyroid glands, consistent with a decreased rate of ⁴⁵Ca release from mucosal cells under the same experimental conditions. Calcium binding to basal-lateral membrane fragments was also sodium dependent and enhanced by the prior administration of parathyroid extract. The consistent correlation between the rate of calcium transport across the basal-lateral membrane of the mucosal cell and the activity of this Na, Ca-dependent phosphatase under a variety of experimental conditions suggest that this enzyme may mediate the parathyroid hormone-sensitive active transport of calcium across the intestine.