The affinities of the (R)‐ and (S)‐enantiomers of hexahydro‐difenidol (1) and its acetylenic analogues hexbutinol (2), hexbutinol methiodide (3) and p‐fluoro‐hexbutinol (4) (stereochemical purity > 99.8%) for muscarinic receptors in rabbit vas deferens (M1), guinea‐pig atria (M2) and guinea‐pig ileum (M3) were measured by dose‐ratio experiments. The (R)‐enantiomers consistently showed higher affinities than the (S)‐isomers. The stereoselectivity ratios [(R)/(S)] were greatest with the enantiomers of 1 (vas deferens: 550; ileum: 191; atria: 17) and least with those of the p‐Fluoro‐analogue 4 (vas deferens: 34; ileum: 8.5; atria: 1.7). The enantiomeric potency ratios for compounds 1–4 were highest in rabbit vas deferens, intermediate in guinea‐pig ileum and much less in guinea‐pig atria. Thus, these ratios may serve as a predictor of muscarinic receptor subtype identity. (S)‐p‐Fluoro‐hexbutinol [(S)‐4] showed a novel receptor selectivity profile with preference for M3 receptors: M3 > M2 ≥ M1. These results do not conform to Pfeiffer's rule that activity differences between enantiomers are greater with more potent compounds.