Soluble guanylyl cyclases are typically obligate heterodimers, composed of a single alpha and a single beta subunit. MsGC-beta3, identified in the tobacco hornworm Manduca sexta, was the first example of a soluble guanylyl cyclase that exhibited enzyme activity without the need for coexpression with additional subunits. Subsequent studies have revealed that the mammalian beta2 subunit also shares this property. Using a combination of gel filtration chromatography, coprecipitation and site-directed mutagenesis we show that, as predicted, MsGC-beta3 forms active homodimers. We also demonstrate that MsGC-beta3 is capable of forming heterodimers with the nitric oxide (NO)-sensitive guanylyl cyclase subunits MsGC-alpha1 and MsGC-beta1. These heterodimers, however, show no enzyme activity and, like mammalian beta2 subunits, act in a dominant negative manner when combined with the NO-sensitive subunits to disrupt their activation by NO. In addition, we show that the unique C-terminal domain of MsGC-beta3 is not necessary for enzyme activity and might act as an auto-inhibitory domain.