Cytochrome P450 2A6 (CYP2A6) is a human enzyme best known for metabolizing tobacco-related compounds, such as nicotine, cotinine (COT), and nitrosamine procarcinogens. CYP2A6 genetic variants have been associated with smoking status, cigarette consumption, and tobacco-related cancers. Our objective was to functionally characterize four nonsynonymous CYP2A6 sequence variants with respect to their haplotype, allele frequency, and association with in vivo CYP2A6 activity. In vivo, nicotine was administered orally to 281 volunteers of Black African descent. Blood samples were collected for kinetic phenotyping and CYP2A6 genotyping. In vitro, nicotine C-oxidation catalytic efficiencies of heterologously expressed variant enzymes were assessed. The four uncharacterized sequence variants were found in seven novel alleles CYP2A6(*)24A&B ; (*)25, (*)26, (*)27, and *28A&B, most were associated with impaired in vivo CYP2A6 activity. Nicotine metabolism groupings, based on the in vivo data of variant alleles, were created. Mean trans-3'-hydroxycotinine/cotinine (3HC/COT) differed (P<0.001) between normal (100%), intermediate (64%), and slow (40%) groups. Systemic exposure to nicotine following oral administration also differed (P<0.001) between normal (100%), intermediate (139%), and slow (162%) metabolism groups. In addition, alleles of individuals with unusual phenotype-genotype relationships were sequenced, resulting in the discovery of five novel uncharacterized alleles and at least one novel duplication allele. A total of 7% of this population of Black African descent had at least one of the eight novel characterized alleles and 29% had at least one previously established allele. These findings are important for increasing the accuracy of association studies between CYP2A6 genotype and behavioral, disease, or pharmacological phenotypes.