In species with external fertilization, the guanylate cyclase family is responsible for the long-distance interaction between gametes, as its activation allows sperm chemotaxis toward egg-derived substances, gamete encounter, and fertilization. In species with internal fertilization, guanylate cyclase-activating substances, which are secreted by several tissues in the genital tracts of both sexes, deeply affect sperm motility, capacitation, and acrosomal reactivity, stimulating sperm metabolism and promoting the ability of the sperm to approach the oocyte, interact with it, and finally fertilize it. A complex system of intracellular pathways is activated by guanylate cyclase agonists in spermatozoa. Sperm motility appears to be affected mainly through an increase in intracellular cAMP, whereas the acrosome reaction depends more directly on cyclic GMP synthesis. Both cyclic nucleotides activate specific kinases and ion signals. A complex cross-talk between cAMP. and cyclic GMP-generating systems occurs, resulting in an upward shift in sperm function. Excessive amounts of certain guanylate cyclase activators might exert opposite, antireproductive effects, increasing the oxidative stress on sperm membranes. In view of the marked influence exerted by guanylate cyclase-activating substances on sperm function, it seems likely that guanylate cyclase activation or inhibition may represent a new approach for the diagnosis and treatment of male and/or female infertility.