This study was designed to examine the in vivo reduction of theN‐oxidation products of nicotine metabolism in rats. Male Fischer‐344 rats were divided into one control and three experimental groups (n = 20). Each treatment group received either 0.02% trans‐nicotineN‘ ‐oxide, 0.02%cis‐nicotineN‘ ‐oxide, or 0.02% nicotineN,N‘‐dioxide in drinking water for 3 wk. After 7 d of metabolite administration, plasma nicotine levels in the trans‐nicotineN‘ ‐oxide group rose to twice that of the cis‐nicotineN‘ ‐oxide or nicotineN,N‘ ‐dioxide group. Plasma cotinine [1 ‐methyl‐5‐(3‐pyridinyl)‐2‐pyrrolidinone] concentrations reached maximum levels during wk 1 in thecis‐nicotineN‘ ‐oxide and nicotineN,N‘ ‐dioxide groups but continued to increase for another 7 d in the trans‐nicotineN‘‐oxide group. At d 15 and again at d 21, rats from each group (n = 10) were placed in metabolism chambers and given 50 ml tap water over a 24‐h period. Analysis of urine obtained from a metabolism‐chamber study conducted after 15 d of consumption revealed concentrations of nicotine in the trans‐nicotineN‘‐oxide group that were 3 times higher than cis‐nicotineN‘‐oxide‐treated animals. Urinary cotinine levels were similar in all three groups. Results from a second chamber study (d 21) showed similar urinary nicotine and cotinine values in all treatment groups. Plasma total triiodothyronine (TT3) concentrations were reduced in all treatment groups during the first week. Plasma total thyronine (TT4) concentrations were reduced (p < 0.05) in the trans‐nicotineN‘‐oxide and cis‐nicotineN‘‐oxide treatment groups during wk 3. An improved method for the preparation synthesis of cis‐nicotineN‘‐oxide is presented. An analytical method for separation of nicotine, cotinine, andcis‐ and trans‐nicotineN‘‐oxide, as well ascis‐ and trans‐nicotineN,N'‐dioxide, is also outlined.