Background and PurposeNitric oxide (NO) is known to activate NO‐sensitive guanylyl cyclase (NO‐GC) and to elicit cGMP production. However, NO has also been proposed to induce cGMP‐independent effects. It is accepted practice to use specific NO‐GC inhibitors, such as ODQ or NS2028, to assess cGMP‐dependent NO effects. Consequently, NO‐induced reactions seen in the presence of these inhibitors commonly serve as an affirmation of cGMP independence.Experimental ApproachWe evaluated the use of ODQ to discriminate between cGMP‐dependent and cGMP‐independent NO effects. NO‐GC‐expressing HEK cells, platelets and tissues from wild type (WT) and NO‐GC‐deficient mice (GCKO) were used.Key ResultsNO donors led to accumulation of cGMP in platelets and GC‐expressing HEK cells and induced phosphorylation of the vasodilator‐stimulated phosphoprotein in platelets; both effects were reduced by ODQ. High concentrations of NO donors, however, overrode the inhibitory effect of ODQ. Correspondingly, ODQ inhibited but did not fully eliminate NO‐induced relaxation of aorta and fundus from WT mice. Relaxation induced by endogenously released NO was fully or partially inhibited by ODQ in fundus and aorta, respectively. In aorta and fundus of GCKO mice NO‐induced relaxation was absent and served as standard for complete NO‐GC inhibition.Conclusions and ImplicationsHigh NO concentrations can overcome the inhibitory effect of ODQ on NO‐GC. Smooth muscle relaxation induced by NO donors/endogenously released NO in the presence of ODQ in WT was absent in GCKO animals indicating involvement of NO‐GC. Accordingly, NO‐induced effects in the presence of ODQ do not necessarily prove cGMP independence.