OBJECTIVE Motilin stimulates gastrointestinal motility, but in vitro studies reveal a direct smooth muscle effect, whereas in vivo studies reveal a neurally mediated effect. The aim of the present study was to determine if motilin binds to microsomes (smooth muscle) and/or synaptosomes (neurons). METHODS Subcellular fractions were prepared from tissue of rabbit gastric antrum and colon by differential centrifugation and density gradient centrifugation and characterized by determining motilin binding and the presence of membrane markers. RESULTS The purified microsomal fraction, enriched in the smooth muscle marker 5'-nucleotidase, was found to have the highest specific motilin binding in both antrum and colon. In the antrum, but not in the colon, the mitochondrial fraction also showed enrichment of [3H]-saxitoxin binding (marker for synaptosomes) and motilin binding, although the latter was much lower than in the microsomal fraction. Two receptor binding sites were characterized in both the antral mitochondrial/synaptosomal and colonic microsomal fraction (antrum: pKd,1 9.89+/-0.19, pKd,2 8.18+/-0.11, colon: pKd,1 9.72+/-0.31, pKd,2 8.39+/-0.58). CONCLUSIONS Motilin binding is predominantly associated with smooth muscle membranes in both antrum and colon of the rabbit. In both organs two motilin binding sites are present with comparable affinities, but the density in the colon is much higher for both sites. Whether they represent neural and smooth muscle receptors will require studies with isolated smooth muscle cells and neurons.