Based on the ability of many receptors to amplify NIH 3T3 cells, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme beta-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. We used this assay to evaluate the pharmacology of agonist and antagonist ligands for five cloned human muscarinic receptor subtypes (m1-m5). When cells were transfected with subtypes that prefer the G-protein Gq (m1, m3, m5) robust increases in enzyme activity were observed. The subtypes that prefer Gi (m2 and m4) only induced beta-galactosidase when co-expressed with a chimera between the G-proteins Gq and Gi (Gq-i5). Overall, the rank-order of potency and intrinsic activity of most of the tested ligands were in remarkably good agreement with earlier results using cloned cell lines and isolated tissues. These data demonstrate that a high throughput colorimetric assay performed in 96-well plates can be used to evaluate subtle differences the pharmacology of ligands for cloned muscarinic receptor subtypes.