The extent to which genetic variation in a population contributes to phenotypic variation depends on the frequency of sequence polymorphisms and the effect of these polymorphisms on function. The frequency of polymorphisms might also reflect the severity of their effects on function. We therefore examined the effect of very rare single nucleotide polymorphisms (SNPs) on the activity of the vesicular monoamine transporter 2 (VMAT2, SLC18A2), a gene implicated in neuropsychiatric disease. Of the two rare SNPs identified in an ethnically diverse population, neither eliminates transport, but one that involves replacement of a highly conserved residue with a very similar amino acid impairs substrate recognition. This variant, and another affecting an unconserved residue, also affect inhibition by the clinically used drug reserpine. Because VMAT2 influences a form of toxicity similar to Parkinson's disease, we extended the analysis to two SNPs identified in a population with Parkinson's disease. These two SNPs have no detectable effect on most aspects of VMAT2 function, but one that affects a highly conserved residue may increase sensitivity to the inhibitor tetrabenazine. The results illustrate the relationship between conservation of the affected residue, the nature of the substitution and effects on substrate versus inhibitor interaction.