1. Physiological nitric oxide (NO) signal transduction occurs through activation of guanylyl cyclase (GC)-coupled receptors, resulting in cGMP accumulation. There are five possible receptors: four heterodimers (alpha1beta1, alpha2beta1, alpha1beta2, alpha2beta2) and a presumed homodimer (nubeta2). The present study investigated the kinetic and pharmacological properties of all these putative receptors expressed in COS-7 (or HeLa) cells. 2. All exhibited NO-activated GC activity, that of alpha1beta1 and alpha2beta1 being much higher than that of the beta2-containing heterodimers or nubeta2. All were highly sensitive NO detectors. Using clamped NO concentrations, EC(50) values were 1 nM for alpha1beta1 and 2 nM for alpha2beta1. With alpha1beta2, alpha2beta2 and nubeta2, the EC(50) was estimated to be lower, about 8 nM. 3. All the GCs displayed a marked desensitising profile of activity. Consistent with this property, the concentration-response curves were bell-shaped, particularly those of the beta2 heterodimers and nubeta2. 4. Confocal microscopy of cells transfected with the fluorescently tagged beta2 subunit suggested targeting to the endoplasmic reticulum through its isoprenylation sequence, but no associated particulate GC activity was detected. 5. The NO-stimulated GC activity of all heterodimers and nubeta2 was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and, except for nubeta2, was enhanced by the allosteric activator YC-1. 6. It is concluded that all the four possible heterodimers, as well as the putative nubeta2 homodimer, can function as high-affinity GC-coupled NO receptors when expressed in cells. They exhibit differences in NO potency, maximal GC activity, desensitisation kinetics and possibly subcellular location but, except for nubeta2, cannot be differentiated using existing pharmacological agents.