Mucosal macrophages play a fundamental role in the regulation of immunological events and inflammation in the small intestine. Because no information is available on normal small intestinal macrophages, we developed a technique for the isolation and purification of jejunal lamina propria macrophages in order to study their phenotype and activity. From sections of normal human jejunum, lamina propria mononuclear cells were isolated by neutral protease digestion and then subjected to counterflow centrifugal elutriation to purify the macrophages. The cells isolated by this procedure contained <1% CD3+ lymphocytes and displayed the size distribution, morphological features, ultrastructure and phagocytic activity of mononuclear phagocytes. In contrast to blood monocytes, however, mucosal macrophages from the jejunum did not exhibit adherence properties or express CD14, a receptor for the lipopolysaccharide-binding protein. The purification of large numbers of lamina propria macrophages by this procedure offers the opportunity to define the role of this cell in the physiological inflammation characteristic of normal intestinal mucosa and the pathological inflammation associated with small intestinal diseases.