1. The acetylcholinesterase inhibitors physostigmine, neostigmine, tetrahydroaminoacridine (tacrine; THA) and diisopropylfluorophosphate (DFP) were tested for possible direct nicotinic actions in rat striatal synaptosomes preloaded with [3H]-dopamine. In this preparation, nicotinic cholinoceptor activation evoked [3H]-dopamine release. 2. Antagonist activity was examined by giving a brief nicotine (1 microM) challenge after 30 min superfusion with an acetylcholinesterase (AChE) inhibitor (0.3-300 microM). Physostigmine, neostigmine and tacrine produced a concentration-dependent blockade. Physostigmine and tacrine were particularly potent (IC50S approx. 10 microM and 1 microM, respectively). DFP reduced nicotinic responses only at the highest concentration tested (300 microM). 3. Nicotinic blockade produced by superfusion with physostigmine (30 microM) was insurmountable when tested against nicotine (0.1-100 microM). 4. Physostigmine (30 microM) also reduced responses to the nicotinic agonists 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and cytisine, but did not alter responses to high K+ or (+)-amphetamine. A higher concentration of physostigmine (300 microM) completely blocked responses to nicotine, somewhat reduced responses to amphetamine, and did not alter responses to high K+. Tacrine (3 microM) reduced responses to nicotine and to high K+ but did not affect responses to amphetamine. 5. Physostigmine (0.3-300 microM), given as a brief pulse, did not produce a nicotinic agonist-like effect. 6. Physostigmine, neostigmine, tacrine and DFP (all at 30 microM) each produced near-total (> 96%) inhibition of AChE activity. However, DFP at a concentration (60 microM) that produced a degree of AChE inhibition equal to that of physostigmine 30 microM, did not significantly reduce nicotine-induced dopamine release. 7. It thus appears that physostigmine blocks CNS nicotinic receptors in an insurmountable and pharmacologically selective manner, independent of its ability to inhibit acetylcholinesterase. Tacrine reduced nicotinic responses, quite possibly by an indirect mechanism. The possibility of direct or indirect blockade of nicotinic receptor-mediated actions may complicate the interpretation of preclinical studies that have employed physostigmine and tacrine.