Cytosolic guanylyl cyclases (GTP pyrophosphate-lyase [cyclizing; EC 4.6.1.2]), primary receptors for nitric oxide (NO) generated by NO synthases, are obligate heterodimers consisting of an alpha and a beta subunit. The alpha1/beta1 form of guanylyl cyclase has the greatest activity and is considered the universal form. An isomer of the beta1 subunit, i.e., beta2, has been detected in the liver and kidney, however, its role is not known. In this study, we investigated the function of beta2. Immunoprecipitation experiments showed that the beta2 subunit forms a heterodimer with the alpha1 subunit. NO-stimulated cGMP formation in COS 7 cells cotransfected with the alpha1 and beta2 subunits was approximately 1/3 of that when alpha1 and beta1 subunits were cotransfected. The beta2 subunit inhibited NO-stimulated activity of the alpha1/beta1 form of guanylyl cyclase and NO-stimulated cGMP formation in cultured smooth muscle cells. Our results provide the first evidence that the beta2 subunit can regulate NO sensitivity of the alpha1/beta1 form of guanylyl cyclase. Northern analysis for guanylyl cyclase subunits was performed on RNA from kidneys of Dahl salt-sensitive rats, which have been shown to have decreased renal sensitivity to NO. Compared to the Dahl salt-resistant rat, message for beta2 was increased, beta1 was decreased, and alpha1 was unchanged. These results suggest a molecular basis for decreased renal guanylyl cyclase activity, i.e. , an increase in the alpha1/beta2 heterodimer, and decrease in the alpha1/beta1 heterodimer.