Previous studies have suggested that in the rat, small intestine is the source for rise in plasma histaminase levels seen after heparin administration. The cellular location of histaminase in intestine and the mechanism of heparin release have not been previously investigated. The present study identifies intestinal villus cells rather than crypt cells as the location of intestinal histaminase; at this site, the enzyme is not associated with brush border. Heparin added to incubations containing isolated intestinal cells did not release histaminase into the medium. Perfusion of intestinal vasculature with heparin caused a prompt release of this enzyme into venous effluent. The present investigation. therefore, suggests that heparin releases histaminase from vascular binding sites rather than directly from parenchymal cells. The use of isolated intestine with perfusion of the vasculature could serve as a useful tool for further defining the relationship between the sites of synthesis and the binding sites involved with heparin releasable enzymes such as histaminase.