Analysis of the characteristics of cerebral binding of [3H] (-)-nicotine revealed a single population of sites with high affinity (KD = 6.0 +/- 0.6 nM). The regional distribution of the binding of [3H] (-)-nicotine was heterogeneous with the largest concentration of binding sites being in the thalamus, cortex and striatum, and a low level of binding activity in the cerebellum and hypothalamus. Competition studies showed that several metabolites and congeners of nicotine potently competed with [3H] (-)-nicotine and their in vitro activity was correlated with behavioural activity, as estimated from previously published data for rats trained to discriminate central effects of nicotine. Conversely, nicotine antagonists, with the exception of dihydro-beta-erythroidine, were weak or inactive in this binding assay. It is concluded that the binding site for [3H] (-)-nicotine investigated probably mediates at least one of the behavioural effects of nicotine, the nicotine discrimative stimulus.