Background: The ratio of two nicotine metabolites, cotinine and trans-3′-hydroxycotinine (3-HC), has been validated as a method of phenotyping the activity of the liver enzyme cytochrome P450 (CYP) 2A6 and, thus, the rate of nicotine metabolism. Our objective was to evaluate the correlates and stability of the 3-HC to cotinine ratio in ad libitum and reducing smokers, using nicotine replacement therapy (NRT), over a period of months. Methods: Smokers (n = 123, 94% Caucasian) participated in a smoking reduction study, where one-third of the sample smoked ad libitum for 8 weeks (Waitlist phase), before joining the rest of the participants for 12 weeks of cigarette reduction (Reduction phase) using NRT. Urinary nicotine, cotinine, and 3-HC were measured at each visit. Results: The baseline 3-HC to cotinine ratio was significantly but weakly correlated with cigarettes per day (r = 0.19), BMI (r = -0.27), and waking at night to smoke (r = 0.23). As assessed by repeated measure ANOVA, the 3-HC to cotinine ratio was stable in the Waitlist phase [coefficient of variation for 3 to 4 measurements, 38% (range, 5-110%)], whereas minor variation was noted in the Reduction phase [coefficient of variation for 3-5 measurements, 35% (range, 10-107%)]. Conclusions: In nonreducing ad libitum smokers, the 3-HC to cotinine ratio was generally stable, whereas during smoking reduction using NRT, some small variation was detected. Although the current findings are suggestive of the stability of the 3-HC to cotinine ratio in a predominantly Caucasian sample smoking freely or reducing smoking with NRT, additional research is needed in more diverse populations.