Soluble guanylyl cyclase (sGC) is composed of one a and one b subunit, each existing in 2 isoforms (a₁/a₂ and b₁/b₂). The aim of our study was to investigate the functional importance of the sGCa₁-subunit in several sGC-mediated vasorelaxations. Therefore, we studied blood vessels from mice lacking the sGCa₁ subunit. From mice of both genders, segments of the thoracic aorta and femoral artery were mounted in a small vessel myograph for isometric tension recording. Concentration-response curves were established on acetylcholine (ACh) (1 nM–10 mM), sodium nitroprusside (SNP) (1 nM–10 mM), NO gas (1 mM–100 mM), BAY 41-2272 (1 nM–10 mM), and levcromakalim (Lev) (1 mM–100 mM) in control conditions and/or in the presence of ODQ. The relaxing influence of endogenous NO (released from the endothelium in response to ACh), exogenous NO (delivered by the NO-donor SNP and NO gas) and BAY 41-2272 (an NO-independent sGC-activator) was significantly reduced in the arteries of the sGCa₁ knock-out mice. However, preparations from sGCa₁ knock-out mice still showed a substantial relaxation in response to exogenous NO and BAY 41-2272. The sGC-inhibitor ODQ strongly diminished the remaining effect of exogenous NO. The sGCa₁ knock-out mice and their wild type littermates showed a similar response to the K_ATP-channel opener Lev, indicating that the reduced NO- and BAY 41-2272-induced responses are not aspecific. All observations were similar in both sexes. Taken together, these findings indicate that the sGCa₁b₁ isoform is involved in the vasorelaxing effect of both endogenous and exogenous NO. However, the substantial relaxation response to exogenous NO still observed in the sGCa₁ knock-out mice suggests the contribution of the sGCa₂b₁ isoform or other ODQ sensitive mechanism.The vasorelaxing effect of BAY 41-2272 in the sGCa₁ knock-out mice, indicates that both sGC isoforms are present in the blood vessels studied.