The cytochrome P450 2A6 (CYP2A6) enzyme metabolizes several clinically relevant substrates, including nicotine—the primary psychoactive component in cigarette smoke. The gene that encodes the CYP2A6 enzyme is highly polymorphic, resulting in extensive interindividual variation in CYP2A6 enzyme activity and the rate of metabolism of nicotine and other CYP2A6 substrates including cotinine, tegafur, letrozole, efavirenz, valproic acid, pilocarpine, artemisinin, artesunate, SM-12502, caffeine, and tyrosol. CYP2A6 expression and activity are also impacted by non-genetic factors, including induction or inhibition by pharmacological, endogenous, and dietary substances, as well as age-related changes, or interactions with other hepatic enzymes, co-enzymes, and co-factors. As variation in CYP2A6 activity is associated with smoking behavior, smoking cessation, tobacco-related lung cancer risk, and with altered metabolism and resulting clinical responses for several therapeutics, CYP2A6 expression and enzyme activity is an important clinical consideration. This review will discuss sources of variation in CYP2A6 enzyme activity, with a focus on the impact of CYP2A6 genetic variation on metabolism of the CYP2A6 substrates.