Since a nitric oxide-sensitive form of guanylyl cyclase exists as a heterodimer, mutations disrupting catalysis but not heterodimer formation could serve as dominant negative mutations. Two mutations within the catalytic region of the alpha subunit (alpha 1D513A, alpha 1D529A) caused complete losses of basal and sodium nitroprusside-stimulated guanylyl cyclase activity; however, the mutant alpha subunits continued to form heterodimers with wild-type beta-subunit. Rat insulinoma cells, which contain the alpha 1 beta 1 form of guanylyl cyclase, were stably transfected with alpha 1D513A or alpha 1D529A. The response to sodium nitroprusside, which exceeded 200-fold in the presence of wild-type alpha 1, was markedly reduced by the expression of either mutant subunit. In contrast, the mutant subunits failed to inhibit heat-stable enterotoxin-induced cGMP elevations; the bacterial peptide elevated insulinoma cell cGMP approximately 100-fold. The two point mutations, therefore, result in dominant negative proteins that can effectively and specifically block the NO/cGMP signaling pathway. These are also the first studies to show that, although both the alpha and beta subunits contain regions homologous to putative cyclase catalytic regions, a point mutation in just one of the subunits can completely inhibit cyclase activity.