As the major effector molecule for NO, soluble guanylyl cyclase (sGC) plays a key role within the NO/cGMP signalling cascade which participates in penile erection. sGC exists as an ab-heterodimer, but only two isoforms have been reported to be active (sGCa₁b₁ and sGCa₂b₁). The a₁b₁ isoform is predominantly present in penile tissue. The functional importance of the a₁-subunit in corpus cavernosum (CC) smooth muscle relaxation was assessed by mounting isolated CC from sGCa₁^-/- mice and wild type littermates in organ baths for isometric tension recording. The endothelium-dependent relaxation to acetylcholine (ACh) and the neurogenic response to electrical field stimulation (EFS) were nearly abolished in the sGCa₁^-/- CC. The relaxing influence of exogenous NO (from sodium nitroprusside (SNP) and NO-gas), was significantly decreased in the sGCa₁^-/- mice. The remaining relaxation induced by exogenous NO in the sGCa₁^-/- mice was strongly but not completely inhibited by the sGC-inhibitor ODQ. In the preparations of the sGCa₁^-/- mice, the response to BAY 41-2272 (NO-independent sGC-activator) was also significantly reduced. The specificity of the impairment of the sGC-related responses was demonstrated by the unaffected response to forskolin (adenylyl cyclase activator) and 8-pCPT-cGMP (cGMP-analogue). In conclusion, our findings indicate the involvement of the predominant isoform sGCa₁b₁ in NO-induced CC smooth muscle relaxation. However, the remaining relaxing influence of exogenous NO in the sGCa₁^-/- mice suggests that also the less abundantly expressed isoform sGCa₂b₁ and/or(an) sGC-independent mechanism(s) has a substantial role in NO-related corporal smooth muscle relaxation.