Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an α and a β subunit. So far, only the α₁β₁ heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the α₁β₁ heterodimer. Here we demonstrate for the first time the natural occurrence of the α₂ subunit on the protein level. The α₂ subunit co-precipitated with the β₁ subunit from human placenta, showing the existence of the α₂β₁ isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the α₂β₁ heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the α₁β₁ form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. ¹H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5´-hydroxymethyl-2´-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration–response curve and turned CO into an effective activator, as it did for the α₁β₁ heterodimer (200-fold activation). In summary, the differences in primary structure of both α subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform.