The location of leucine beta-naphthylamidase on the outer surface of the microvillous membrane of rabbit small intestine was examined by analyzing the interaction of antibodies against leucine beta-naphthylamidase or another microvillous enzyme, sucrase-isomaltase complex, with microvillous vesicles having different relative amounts of these enzymes, in respect to vesicle agglutination, inhibition of enzyme activity, and electron-microscopic morphology. The results obtained indicate that leucine beta-naphthylamidase, or at least its antigenic sites, protrude about 10 nm from the outer surface of the microvillous membrane.