Passage of spermatozoa through the epididymis is obligatory for sperm maturation processes and is based on spontaneous phasic contractions (SC) of the epididymal duct. Here, the functional role of cyclic GMP (cGMP) signaling in modulating SC in the bovine epididymal caput and corpus region was examined by muscle tension recording, immunological and autoradiographic techniques. The cGMP-analog 8-Br-cGMP as well as the nitric oxide (NO) donor sodium nitroprusside (SNP) and the natriuretic peptides (NPs) atrial NP (ANP) and C-type NP (CNP) displayed distally increasing SC-relaxant effects. In agreement, a distally increasing epididymal expression of the cGMP-dependent protein kinase I (PKG I), endothelial NO synthase (eNOS) and the ANP receptor was found. Immunoreactivity for PKG, soluble guanylate cyclase (sGC) and eNOS could be localized to the epididymal muscle cells as well as to the epithelial basal cells only at the corpus level. The SC-relevant action of NO and the NPs was cGMP-dependent, and the action of 8-Br-cGMP, in turn, was modified by epithelial and luminal factors. The NOS inhibitor L-NAME caused an increase in SC frequency, indicating basal activity of NO generating enzymes. The SC-inhibitory effect of 8-Br-cGMP was clearly reduced by the PKG inhibitor Rp-8-Br-cGMPS as well as by iberiotoxin, thapsigargin and indomethacin, pointing to PKG as main SC-relevant target of cGMP, and to BK channels, the sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase and cyclooxygenase-1 as possible targets of PKG. These data support an essential role of cGMP signaling in the control of epididymal peristalsis, thereby enabling fine tuning of sperm transport and maturation.