1. The effect of acute and chronic (16 days) administration of nicotine on cortical acetylcholine (ACh) release, gross behaviour and brain nicotinic binding sites was investigated in rats and guinea-pigs. 2. The drug, injected either subcutaneously (0.45-0.90 mg kg-1) or intracerebroventricularly (1, 3 and 5 micrograms) increased the cortical ACh release, in a dose-dependent manner, through mecamylamine-sensitive receptors for 1-2 h in both species. 3. Chronic treatment significantly increased basal ACh release in the rat and slightly lowered it in the guinea-pig, but the response to a challenging dose of nicotine was proportionally maintained in both species. 4. The number of nicotinic receptors was four times higher in the rat than in the guinea-pig and was not dependent on the radioligand used ([3H]-nicotine or [3H]-ACh, in the presence of atropine) to determine this. The nicotinic binding sites showed an apparent increase in chronically treated rats but no change in guinea-pigs. 5. Tolerance to the inhibitory effect of the drug, assessed with the T maze test, was found in the rat. No apparent change in gross behaviour was detected in the guinea-pig. 6. It is concluded that chronic nicotine treatment causes evident tolerance to its inhibitory effect on behaviour in the rat, but no adaptation to its excitatory properties on the cholinergic brain structures in rats and guinea-pigs.