Polyclonal antipeptide antibodies have been raised against each of the two isoforms of the rat vesicular monoamine transporter, VMAT1 and VMAT2. Antibody specificity was determined by isoform-specific staining of monkey fibroblasts programmed to express either VMAT1 or VMAT2. The expression of VMAT1 and VMAT2 in the diffuse neuroendocrine system of the rat has been examined using these polyclonal antibodies specific for either VMAT1 or VMAT2. VMAT1 is expressed exclusively in endocrine/paracrine cells associated with the intestine, stomach, and sympathetic nervous system. VMAT2 is expressed in neurons of the sympathetic nervous system, and aminergic neurons in the enteric and central nervous systems. VMAT2 is expressed in at least two endocrine cell populations in addition to its expression in neurons. A subpopulation of chromogranin A (CGA)-expressing chromaffin cells of the adrenal medulla also express VMAT2, and the oxyntic mucosa of the stomach contains a prominent population of CGA- and VMAT2-positive endocrine cells. The expression of VMAT2 in neurons, and the mutually exclusive expression of VMAT1 and VMAT2 in endocrine/paracrine cell populations of stomach, intestine, and sympathetic nervous system may provide a marker for, and insight into, the ontogeny and monoamine-secreting capabilities of multiple neuroendocrine sublineages in the diffuse neuroendocrine system.