Histamine mediates signalling by a wide range of neural and non-neural cells including mast cells. Like other biogenic amines, histamine is released from specialized secretory vesicles and requires transport from the cytoplasm into these vesicles. Of the two vesicular monoamine transporters, histamine potently inhibits 3H-serotonin transport by one (VMAT2) but not the other (VMAT1). In addition, histamine-containing cells in both neural and non-neural cells express VMAT2. However, histamine lacks the hydroxyl groups generally considered necessary for recognition as a substrate by the vesicular monoamine transporters. Using a heterologous expression system, we now report that VMAT2 not only shows inhibition by histamine but also transports 3H-histamine. Interestingly, histamine differs from other monoamine transmitters and does not inhibit 3H-reserpine binding to VMAT2, indicating interaction at a distinct site. Surprisingly, reserpine inhibits histamine transport with much less potency than serotonin transport, suggesting a different transport mechanism. However, replacement of serines in the third transmembrane domain of VMAT2 that have been shown to be essential for recognition of other monoamines also eliminate 3H-histamine transport, suggesting that these serine residues may do more than simply recognize the hydroxyl groups on a monoamine substrate.