In the present study, lobeline and two structurally simplified analogs were evaluated for activity in muscarinic and nicotinic binding assays, a functional assay for nicotinic receptor activation (86Rb+ efflux from striatal synaptosomes) and an acetylcholinesterase (AChE) assay. Lobeline displaced [3H]cytisine binding to rat cortical membranes with a mean inhibition constant (KI) value of 16.0 nM, while the lobeline analogs CRM-I-13-1 and CRM-I-32-1 exhibited values of 15.0 and 5.4 microM, respectively. [3H]methylscopolamine was displaced by lobeline with a mean KI value of 37.0 microM while CRM-I-13-1 and CRM-I-32-1 exhibited values of 55.0 and 16.0 microM, respectively. While nicotine stimulated 86Rb+ efflux from striatal synaptosomes in a mecamylamine reversible manner at each concentration tested, lobeline slightly increased 86Rb+ efflux at lower concentrations and reduced efflux at higher concentrations. Further, none of the lobeline effects were reversed with mecamylamine. Although less potent, the two lobeline analogs exhibited a similar pattern of activity. These data may suggest that lobeline and structurally similar compounds bind with different subtype selectivity than nicotine, or exert their agonists effects through non-nicotinic mechanisms. All of the compounds tested were at least several hundred times less potent than physostigmine as AChE inhibitors. While some differences were apparent between the lobeline analog which contained the 2-keto-ethyl portion of lobeline and the analog which contained the phenyl 2-hydroxy-ethyl moiety, each compound was much less active than lobeline in most parameters assessed.