A new radiometric assay for nicotine and 12 of its metabolites disclosed that plasma nicotine and cotinine t1/2 beta were independent of dose after single intraarterial nicotine doses of 0.1, 0.5, or 1.0 mg/kg. At high doses, nicotine AUC and clearance tended to exhibit a small degree of dose dependency. The longest lived metabolites, cotinine-N-oxide and a previously unidentified metabolite now revealed to be allohydroxydemethylcotinine, persisted for 96 hr after nicotine injection, whereas cotinine was detected for only 48 hr. Cotinine, formerly considered the longest lived nicotine metabolite, serves widely as the most sensitive indicator of prior exposure to small concentrations of nicotine. The present studies disclose new, longer lasting metabolites that may perform this function more sensitively, at least in the rat. At the 3 doses of nicotine administered, plasma nicotine half-life ranged from 0.9 to 1.1 hr; total body clearance of nicotine ranged from 2.9 to 3.9 liters.hr-1.kg-1; and apparent volume of distribution of nicotine from 4.7 to 5.7 liters.kg-1. Also at these 3 doses, mean half-lives of urinary excretion of cotinine, cotinine-N-oxide, and allohydroxydemethylcotinine ranged from 4.8 to 5.3 hr, from 7.9 to 8.2 hr, and from 9.9 to 11.0 hr, respectively.