Our aim was to study whether nitric oxide (NO) donor-induced inhibition of pregnant rat myometrium contractility correlates with the release of NO. Uterine rings from mid-pregnant and late pregnant Sprague-Dawley rats were used for isometric tension recording. Concentration-response relationships to sodium nitroprusside (SNP), nitroglycerine (NTG) and diethylamine (DEA)/NO were assessed. The time course of NO release after addition to the organ chambers of the 3 NO-donors was assessed by the detection of NO products NOx (NO3+NO2) using the microdialysis probe by a HPLC-NO detector system. DEA/ NO induced greater inhibition of the spontaneous contractile activity of uterine rings from mid-pregnant rats than SNP or NTG. In uterine rings from late pregnant rats, however, the maximal inhibition of the contractility by all 3 NO-donors were significantly less. The NOx levels measured in the uterine ring walls from either mid-pregnant or late pregnant rats significantly increased after DEA/ NO as compared to the basal levels or the levels after NTG or SNP. The decrease of NO-donor-induced inhibition of rat myometrium contractility, with unchanged formation of NOx, at term, suggests that the changes in NO signaling are responsible for gestational age-dependent attenuation of the inhibitory effect.