Background Nicotine and a variety of other drugs and toxins are metabolized by cytochrome P450 (CYP) 2A6. Our objective was to evaluate the use of oral nicotine with measurement of the trans‐3′‐hydroxycotinine (3HC)/cotinine (COT) metabolite ratio as a noninvasive probe of CYP2A6 activity. Methods Sixty‐two healthy volunteers received an oral solution of deuterium‐labeled nicotine (2 mg) and its metabolite cotinine (10 mg). Plasma nicotine and plasma and saliva cotinine and 3HC concentrations were measured over time. Results The 3HC/COT ratio derived from deuterium–labeled cotinine, measured in either plasma (2–8 hours after administration) or saliva (at 6 hours), was strongly correlated with the oral clearance of nicotine (r = 0.76–0.83, depending on the time of measurement). The 6–hour 3HC/COT ratio from nicotine derived from tobacco in 14 smokers was highly correlated with the ratio derived from deuterium–labeled nicotine (r = 0.88) and was also highly correlated with the oral clearance of nicotine (r = 0.90). Two subjects homozygous for inactive CYP2A6 alleles produced no 3HC, confirming the specificity of the metabolite ratio. The 3HC/COT ratio was also highly correlated with the clearance and half–life of cotinine, consistent with the fact that cotinine is also primarily metabolized by CYP2A6. Conclusions The 3HC/COT ratio derived from nicotine either administered as a probe drug or from tobacco use, measured in either plasma or saliva, is highly correlated with the oral clearance of nicotine. The ratio appears to be a useful noninvasive marker of the rate of nicotine metabolism (which is important in studying nicotine addiction and smoking behavior), as well as a general marker of CYP2A6 activity (which is important in studying drug and toxin metabolism). Clinical Pharmacology & Therapeutics (2004) 76, 64–72; doi: 10.1016/j.clpt.2004.02.011