Ca2+-ATPase cytochemistry frequently uses the incubation medium of Ando et al. that was introduced in 1981. Some studies, however, have suggested that this medium localizes ecto-ATPase in addition to Ca2+-ATPase and that Ca2+-ATPase is sensitive to fixation. Strong activity of the enzyme on the luminal surface of the blood-brain barrier (BBB) also is considered indicative of immature or pathological microvessels. We address here five questions. 1) Is the incubation medium of Ando et al. specific for BBB Ca2+-ATPase or does it also localize ecto-ATPase? 2) How are the two enzymes distributed in the BBB? 3) How would data interpretation be prone to error if the cytochemical study does not use controls identifying ecto-ATPase? 4) Does the amount of reaction product of both enzymes vary significantly when the cortical tissue is exposed to different fixatives? 5) Does the presence of Ca2+-ATPase on the luminal membrane of the BBB necessarily indicate immature or abnormal brain endothelial cells? Adult male Sprague-Dawley rats were perfused with one of two different fixatives and vibratome slices of the brain cortex were incubated in the medium of Ando et al. The controls used were those demonstrating the ecto-ATPase and those that do not. The results indicate that the incubation medium is not specific for Ca2+-ATPase, because it also localizes the ecto-ATPase. Ca2+-ATPase appears to be localized primarily on the luminal surface of the BBB, while ecto-ATPase is localized on both the luminal and abluminal surfaces. The portion of the reaction product contributed by Ca2+-ATPase would not have been identified if the controls uniquely identifying the ecto-ATPase had not been used. The amount of reaction product formed by Ca2+-ATPase is strongly dependent on the type of fixative used. The strong localization of Ca2+-ATPase on the luminal surface of the BBB is not only normal, but also better accounts for the physiological homeostasis of Ca2+ across the blood-brain interface and should not be interpreted as indicative of immature or pathological microvessels.