RS-42358-197[(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1 H-benzo[de]isoquinolin-1-one hydrochloride] displaced the prototypic 5-hydroxytryptamine3 (5-HT3) receptor ligand [3H]quipazine in rat cerebral cortical membranes with an affinity (pKi) of 9.8 +/- 0.1, while having weak affinity (pKi < 6.0) in 23 other receptor binding assays. [3H]RS-42358-197 was then utilized to label 5-HT3 receptors in a variety of tissues. [3H]RS-42358-197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108-15 cells, and rabbit ileal myenteric plexus with affinities (KD) of 0.12 +/- 0.01, 0.20 +/- 0.01, and 0.10 +/- 0.01 nM and densities (Bmax) of 16.0 +/- 2.0, 660 +/- 74, and 88 +/- 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [3H]RS-42358-197 was similar to that labelled with [3H]GR 65630, but was significantly less than that found with [3H]-quipazine. The binding of [3H]RS-42358-197 had a pharmacological profile similar to that of [3H]quipazine, as indicated by the rank order of displacement potencies: RS-42358-197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT3 receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [3H]RS-42358-197 binding. [3H]RS-42358-197 also labelled a population (Bmax = 91 +/- 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (KD = 1.6 +/- 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT3 receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [3H]RS-42358-197 have detected differences in 5-HT3 receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT3 receptor.