Intestinal brush borders prepared from vitamin D-deficient rats demonstrate increased susceptibility in vitro to fragmentation by shear forces or to loss of microvillus enzymes on treatment with EDTA. These effects are relatively nonspecific and are also observed in normal rats starved for 48 h. They may underlie prior observations that purport to demonstrate a vitamin D-dependent increase in brush border Ca-dependent ATPase. In addition, however, vitamin D increases ATPase activity dependent on certain divalent cations, including Ca and Zn, in whole-particulate suspensions pelleted by high-speed centrifugation of mucosal homogenates. This action is independent of changes in other microvillus enzymes, i.e. disaccharidases, and tissue distribution and cation specificity studies support the hypothesis that the mucosal whole-particulate ATPase is related to transport of Ca, Zn, and possibly other divalent cations.