Of the two active soluble guanylyl cyclase (sGC) isoforms (a₁b₁ anda₂b₁)_, the a₁b₁ isoform is predominantly present in vascular tissue and is therefore believed to play a dominant role in vasorelaxation. This was investigated on aortic and femoral artery segments isolated from sGCa₁^-/- mice and their wild type littermates and by measuring of the cGMP level and sGC enzyme activity. The functional importance of sGCa₁b₁ was demonstrated by a significantly reduced response to acetylcholine, sodium nitroprusside (SNP), NO-gas, YC-1 and BAY 41-2272 in arteries of sGCa₁^-/- mice. However, those substances still had a substantial relaxing effect in these arteries, indicating that not only sGCa₁b₁ is involved in vasorelaxation. The non-upregulation of the sGCa₂ gene and the non-significant increase in cGMP-level in response to SNP, do not support the involvement of the minor sGCa₂b₁ isoform and thus rather suggest (an) sGC-independent mechanism(s). The similarity in the response to the cGMP-analogue 8-pCPT-cGMP between wild type and sGCa₁^-/- mice, indicates that the sGCa₁^-/- mice are not more sensitive towards cGMP. The response to the phosphodiesterase type-5 inhibitor, T-1032 was nearly abolished in the arteries of the sGCa₁^-/- mice, indicating that in those mice there is no accumulation of basal cGMP produced by sGCa₂b_1. Again those findings are against the importance of sGCa₂b_1. On the other hand,the inhibition of the nitric oxide-induced relaxation and cGMP production in the sGCa₁^-/- mice by ODQ suggest that sGCa₂b₁ is functionally active_. This is also suggested from the significant increase in sGC activity in the sGCa₁^-/- mice after addition of BAY-41-2272. It is concluded that besides sGCa₁b_1, also sGCa₂b₁ and/or(an) sGC-independent mechanism(s) has a substantial role in nitric oxide-related vasorelaxation.