Nicotine enhances cognitive performance, and in the zebra finch (Taeniopygia guttata), which is a well-established model of cognition, the effect of nicotine on song production has been reported. Nicotine and cotinine plasma levels were assessed in vivo after s.c. injection of 0.18 mg/kg nicotine, a dose that elicits changes in song production. The half-life of nicotine elimination was 33 min, and levels were undetectable by 4h. Average plasma nicotine over 2 hours was 32 ng/ml, similar to levels seen in human smokers and rat models of nicotine behavior. Nicotine brain levels were 30 and 14 ng/g at 1 and 2 h after treatment. To understand the potential for drug interactions and regulation of nicotine metabolism in zebra finches, we characterized in vitro nicotine metabolism and the hepatic enzyme involved. In humans, cytochrome P450 (CYP) 2A6 metabolizes nicotine to cotinine, and CYP2A-like activity and protein have been reported in some birds. Zebra finch liver microsomes metabolized nicotine and bupropion (CYP2B substrate) but not coumarin (CYP2A substrate). Nicotine was metabolized to cotinine with a Km of 96 μM and Vmax of 56 pmol/min/mg. Nicotine and bupropion metabolism were inhibited by C-8-xanthate (specific CYP2B inhibitor) but not CYP2A-specific inhibitors, and hepatic levels of CYP2B-like but not CYP2A-like proteins correlated with nicotine (r = 0.52, p = 0.04) and bupropion metabolism (r = 0.81, p < 0.001), suggesting CYP2B-mediation of nicotine metabolism as seen in rats. These results will facilitate further investigation of nicotine's effects in zebra finches.