Soluble guanylyl cyclase (SGC) is the main receptor for the gaseous signalling molecule nitric oxide (NO) in vertebrates and invertebrates. Recently, a novel class of drugs that regulate mammalian SGC by NO-independent allosteric mechanisms has been identified [e.g. 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, YC-1]. To assess the evolutionary conservation and hence the potential physiological relevance of these mechanisms, we have tested YC-1 on the brains of two model insects, the cockroach Periplaneta americana and the locust Schistocerca gregaria. YC-1 strongly potentiated the NO-induced elevation of total cyclic 3',5'-guanosine monophosphate (cGMP) and amplified the intensity and consistency of NO-induced cGMP-immunoreactivity in the brain. Our data indicate that the effect of YC-1 was independent of phosphodiesterase inhibition and thus mediated by direct sensitization of SGC. Immunohistopharmacology and co-labelling with antibodies against the SGC alpha-subunit confirmed that cGMP induced by co-application of NO and YC-1 is predominantly attributable to SGC. The staggering number of NO-responsive neurons revealed by YC-1 suggests that previous studies may have considerably underestimated the number of cellular targets for NO in the insect brain. Moreover, a subset of these targets exhibited cGMP-immunoreactivity without application of exogenous NO, demonstrating that YC-1 can be exploited for visualization of physiological cGMP signals in response to endogenous NO production. In conclusion, our discovery that YC-1 is a potent sensitizer of insect SGC indicates that a NO-independent regulatory site is an evolutionarily conserved feature of SGC. Our findings add considerable momentum to the concept of an as yet unidentified endogenous ligand that regulates the gain of the NO-cGMP signalling pathway.