R-(+)-hyoscyamine, the dextro enantiomer of atropine, has been shown to amplify cholinergic transmission. R-(+)-hyoscyamine, unlike S-(—)-hyoscyamine, was able to increase acetylcholine release both in vitro and in vivo at a range of concentrations (10 −14 to 10 −12 M) and doses (5 μg/kg i.p.) which were inadequate for blocking muscarinic receptors. The increase over control values in ACh release was 15.9 ± 2.1% in in vitro experiments performed in rat phrenic nerve-hemidiaphragm preparations ( n = 6), and 63.3 + 16.3% in cortical microdialysis performed in free-moving rats ( n = 5). The maximum ACh release was reached 60 min after R-(+)-hyoscyamine administration in in vivo experiments. At the same doses and concentrations, R-(+)-hyoscyamine was also able to elicit: antinociception of a cholinergic type (55.6–112.7% depending on the test used); complete prevention of scopolamine- and dicyclomine-induced amnesia; potentiation of muscular contractions electrically evoked in isolated guinea-pig ileum (16.7 ± 3.6%) and in rat phrenic nerve-hemidiaphragm (19.9 ± 3.2%) preparations. Antinociception was performed using the hot-plate and acetic acid abdominal constriction tests in mice, and the paw pressure test in rats, while prevention of induced amnesia was evaluated in mice using the passive-avoidance test. The respective affinities (p A 2) for R-(+)- and S-(—)-hyoscyamine vs M 1 (rabbit vas deferens), M 2 (rat atrium) and M 3 (rat ileum) receptor subtypes were as follows: 7.05 ± 0.05/9.33 ± 0.03 for M 1; 7.25 ± 0.04/8.95 ± 0.01 for M 2; 6.88 ± 0.05/9.04 ± 0.03 for M 3. The respective p K i values for R-(+)- and S-(—)-hyoscyamine vs the five human muscarinic receptor subtypes expressed in Chinese hamster oocytes (CHO-K 1) were as follows: 8.21 ± 0.07/9.48 ± 0.18 for m 1; 7.89 ± 0.06/9.45 ± 0.31 for m 2; 8.06 ± 0.18/9.30 ± 0.19 for m 3; 8.35 ± 0.11/9.55 ± 0.13 for m 4; 8.17 ± 0.08/9.24 ± 0.30 for m 5. © 1997 Elsevier Science Ltd. All rights reserved.