We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes M1, M3 or M4. Here we describe allosteric effects at human M1-M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at M1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at M1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPgammaS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect.