Muscarinic activation of bovine tracheal smooth muscle (BTSM) is involved in cyclic guanosine monophosphate (cGMP) production mediated through soluble (sGC) and membrane-bound (mGC) guanylyl cyclases. A muscarinic- and NaCl-sensitive mGC exists in BTSM regulated by muscarinic receptors coupled to G proteins. To identify the mGCs expressed in BTSM, reverse transcriptase/polymerase chain reaction (RT-PCR) from total RNA was performed using degenerate oligonucleotides for amplification of a region conserved among GC catalytic domains. Cloning of amplification products revealed that 76% of all BTSM GC transcripts corresponded to the sGC beta1 subunit and 24% to the B-type (C-type NP 1-22 [CNP]-sensitive) GC receptor. cGMP production by BTSM membrane and soluble fractions confirmed that sGC activity is 3-fold with respect to mGC activity. RT-PCR using specific oligonucleotides revealed that A (atrial NP-sensitive) and C (guanylin-sensitive) mGC subtypes are also expressed in BTSM. Stimulation of basal plasma membrane GC activity by CNP was higher than that by ANP, whereas guanylin showed no effect, indicating that CNP-sensitive guanylyl cyclase (GC-B) is the predominant functional BTSM mGC subtype. Strong adenosine triphosphate inhibition of CNP-stimulated mGC activity supports the finding that the tracheal mGC isoform belongs to the natriuretic peptide-sensitive mGCs. Additionally, CNP was able to reverse the chloride inhibition of BTSM mGC activity, suggesting that this is a novel G protein-coupled GC-B receptor.