Several behavioral studies report that adolescent rats display a preference for nicotine compared with adults. However, age-related pharmacokinetic differences may confound the interpretation of these findings. Thus, differences in pharmacokinetic analyses of nicotine were investigated. Nicotine was administered via acute s.c. (1.0 mg base/kg) or i.v. (0.2 mg base/kg) injection to early adolescent (EA; postnatal day 25) and adult (AD; postnatal day 71) male Wistar rats. Nicotine and its primary metabolite, cotinine, and additional metabolites nornicotine, nicotine-19-N-oxide, trans-39-hydroxycotinine, and norcotinine were sampled from 10 minutes to 8 hours (plasma) and 2 to 8 hours (brain) post nicotine and analyzed by liquid chromatography-tandem mass spectrometry. Following s.c. nicotine, the EA cohort had lower levels of plasma nicotine, cotinine, and nicotine-19-N-oxide at multiple time points, resulting in a lower area under the plasma concentration-time curve (AUC) for nicotine (P < 0.001), cotinine (P < 0.01), and nicotine-19-Noxide (P < 0.001). Brain levels were also lower for these compounds. In contrast, the EA cohort had higher plasma and brain AUCs (P < 0.001) for the minor metabolite nornicotine. Brain-to-plasma ratios varied for nicotine and its metabolites, and by age. Following i.v. nicotine administration, similar age-related differences were observed, and this route allowed detection of a 1.6-fold-larger volume of distribution and 2-fold higher plasma clearance in the EA cohort compared with the AD cohort. Thus, unlike in humans, there are substantial age differences in nicotine pharmacokinetics such that for a given nicotine dose, adolescent rats will have lower plasma and brain nicotine compared with adults, suggesting that this should be considered when interpreting animal model data.