C57BL/6 male mice were injected intraperitoneally with nicotine (2.0 mg/kg) or saline three times each day (0800 h, 1300 h and 1800 h) for a period of 12 days and then tested for nicotine tolerance using a series of behavioral and physiological tests. For each of these tests, animals that received chronic nicotine treatment were significantly less sensitive to nicotine challenge than were animals that received chronic saline treatment, as indicated by shifts to the right of dose-response curves. Animals were retested for nicotine sensitivity 2 weeks following cessation of chronic nicotine injections. Tolerance to acute nicotine challenge persisted in nicotine-treated animals. Chronic nicotine treatment by injections did not alter the binding of L-[3H]-nicotine or alpha-[125I]-bungarotoxin in any of eight brain regions. Plasma corticosterone (CCS) levels were determined in animals prior to the initiation of the injection series (day 0), and on days 4, 8 and 12 of chronic treatment, immediately before the first injection of the day. CCS levels in nicotine-treated animals were elevated as compared to saline-injected controls by day 12 of treatment. Nicotine-treated animals also had elevated CCS levels 2 weeks after the last chronic injection. Nicotine-treated animals were, however, tolerant to nicotine-induced CCS release. Since previous studies from our laboratory have demonstrated that plasma CCS levels are inversely correlated with sensitivity to nicotine, it is possible that the tolerance to nicotine measured following chronic treatment by injections is due, at least in part, to the elevation in plasma CCS levels.