The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.