Myocardial capillary endothelial cells, arteriolar endothelial cells, and the arterial adventitia show positive alkaline phosphatase (AP) enzyme reaction and immunoreactivity in both rat and human heatts. In guinea pigs, however, capillary endothelial staining is discontinuous and arterial adventitia is negative. The ultrastructural correlate of discontinuous capillary staining is a pronounced labeling of pericytes in guinea pig heart and relatively weak endothelial staining. In rat and human heart, enzyme reaction products are localized mainly on plasma membranes and cytotic vesicles of endothelial cells.Comparison of two strains of rat reveals a more dense deposition of enzyme reaction product along the luminal and particularly along the abluminal plasma membrane of SpragueDawley rats than of Wistar rats. Quantitative analysis of immunogold labeled anti-AP antibody density confiims the pronounced polarity of capillary endothelial cell labeling in Sprague-Dawley rats. More than 80% of total endothelial AP protein in Sprague-Dawley rats is localized over the abluminal plasma membrane and basal lamina, as compared with less than 30% in Wistar rats. Moreover, the total endothelial cell labeling is almost sixfold higher in SpragueDawley than in Wistar rats. Total endothelial labeling and proportion of labeling on the abluminal endothelial plasma membrane in human hearts is intermediate between the two strains of rat. The strain and species differences in enzyme distribution could provide important information concerning enzyme function. (JHstochem Cytochem 41:1813-1821, 1993)