Catecholamines are accumulated in vesicles by a proton gradient-dependent transport, which has mostly been studied in bovine chromaffin granules. The full sequence of a cDNA encoding a vesicular transporter from bovine chromaffin cells, bVMAT2, was recently reported. We now present an analysis of bVMAT2, expressed in transfected COS cells. Comparing the binding of a labelled ligand, [3H]TBZOH, and the rate of uptake, we find a much lower molecular turnover number than in chromaffin granules, probably indicating that a majority of expressed transporters are correctly folded and possess the ligand binding site but cannot actively transport monoamines because they are located in compartments which do not possess a proton gradient. The substrate specificity of uptake and its pharmacological sensitivity to various inhibitors closely resemble those previously observed in chromaffin granules. These results suggest that VMAT2 is the major transporter in bovine adrenal glands, and raise the question of the significance of the second related transporter, VMAT1, which is also expressed in this tissue.