Background: At therapeutic concentrations, cytochrome P4502B6 (CYP2B6) is the major P450 isoform catalyzing hepatic ketamine N-demethylation to norketamine in vitro. The CYP2B6 gene is highly polymorphic. The most common variant allele, CYP2B6∗6, is associated with diminished hepatic CYP2B6 expression and catalytic activity compared with wild-type CYP2B6∗1/∗1. CYP2B6.6, the protein encoded by the CYP2B6∗6 allele, and liver microsomes from CYP2B6∗6 carriers had diminished ketamine metabolism in vitro. This investigation tested whether humans with the CYP2B6∗6 allele would have decreased clinical ketamine metabolism and clearance. Methods: Thirty volunteers with CYP2B6∗1/∗1, ∗1/∗6, or ∗6/∗6 genotypes (n = 10 each) received a subsedating dose of oral ketamine. Plasma and urine concentrations of ketamine and the major CYP2B6-dependent metabolites were determined by mass spectrometry. Subjects' self-assessment of ketamine effects were also recorded. The primary outcome was ketamine N-demethylation, measured as the plasma norketamine/ketamine area under the curve ratio. Secondary outcomes included plasma ketamine enantiomer and metabolite area under the plasma concentration-time curve, maximum concentrations, apparent oral clearance, and metabolite formation clearances. Results: There was no significant difference between CYP2B6 genotypes in ketamine metabolism or any of the secondary outcome measures. Subjective self-assessment did reveal some differences in energy and level of awareness among subjects. Conclusions: These results show that while the CYP2B6∗6 polymorphism results in diminished ketamine metabolism in vitro, this allelic variant did not affect single, low-dose ketamine metabolism, clearance, and pharmacokinetics in vivo. While in vitro drug metabolism studies may be informative, clinical investigations in general are needed to validate in vitro observations.