The influence of several imidazolines and sigma-site ligands on cation influx through the 5-HT3 receptor channel in N1E-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [14C] guanidinium induced by 1 microM 5-HT (in the presence of 10 microM substance P in all experiments). In addition, we determined specific binding of [3H]DTG (1,3-di(2-tolyl)-guanidine), a selective sigma-site radioligand, and [3H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1- propanone), a selective 5-HT3 receptor antagonist, to membranes prepared from N1E-115 cells. The 5-HT-induced [14C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT3 receptor channel was mimicked by the sigma-site ligands, (+/-)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine). haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcaine and spermidine.-Ondansetron inhibited [3H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT3 receptor by antazoline, BDF 6143, idazoxan, cirazoline, (+/-)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [3H]DTG, (+/-)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (Ki values in the range of 1 microM or lower), whereas ondansetron, antazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity, in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC50% values) in inhibiting 5-HT-induced [14C]guanidinium influx with their affinities (pKi values) at the 5-HT recognition sites of the 5-HT3 receptor and at the sigma 2-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites. In conclusion, our data suggest that imidazolines and sigma-ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT3 receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT3 receptor channels, at least in part, by interacting with sigma 2-binding sites.