Objective: The aim of the study was to investigate the in vitro (on tension) and in vivo (on intrauterine pressure) effects of a spontaneously releasing nitric oxide adduct, diethylenetriamine–nitric oxide, on rat uterine contractility. Study design: Contractile responses to the nitric oxide donor diethylenetriamine–nitric oxide on isometric tension of rat uterine strips (in vitro) and on intrauterine pressure (in vivo) in anesthetized and conscious animals were quantified at late gestation and during preterm and term labor. Preterm labor was induced with the administration of a single injection of the antiprogestin onapristone (10 mg). All control animals were injected with diethylenetriamine, the parent compound, without nitric oxide. Results: The nitric oxide donor diethylenetriamine–nitric oxide relaxes rat uterine tissues when given in vitro during gestation (median effective dose 0.30 ± 0.09 mmol/L) but fails to have an effect on uterine tissues from laboring term and preterm animals in the muscle bath. Intraperitoneal injection of the nitric oxide adduct in doses of 5 mg and 50 mg produced a significant and sustained decrease in intrauterine pressure in both delivering and nondelivering animals compared with intrauterine pressure values before administration. Analysis of intrauterine pressure levels at different time frames (before injection, immediately after injection, and after 30, 60, and 90 minutes) in anesthetized rats between days 19 and 21 of gestation shows that the dose of 5 mg diethylenetriamine–nitric oxide significantly ( p < 0.05) decreased contractility starling at 30 minutes after administration. Similarly, during labor diethylenetriamine–nitric oxide (5 mg intraperitoneally) decreased contractility ( p < 0.05) but only starting at 60 minutes after injection. However, with the dose of 50 mg diethylenetriamine–nitric oxide contractility was reduced 30 minutes ( p < 0.05) after treatment during pregnancy (days 19 to 21). The same dose of diethylenetriamine–nitric oxide produced a greater relaxation immediately after intraperitoneal injection ( p < 0.05) in the laboring group. Diethylenetriamine–nitric oxide (50 mg) also induced an earlier onset and greater relaxation in animals delivering preterm compared with nondelivering animals at the same stage of gestation. The intrauterine pressure maintained constant contractility levels throughout the 100-minute recording period in the control groups of anesthetized and conscious animals that were injected with diethylenetriamine at the equivalent doses. In the preterm laboring group the effects were similar in both anesthetized and conscious animals. Conclusion: Although in vitro studies of uterine muscle show decreased inhibitory responses to nitric oxide donors during spontaneous term labor and preterm labor compared with tissues collected during late pregnancy (day 19), studies in vivo demonstrate that nitric oxide donors can decrease uterine contractility even more effectively during delivery. The results suggest that nitric oxide donors may act indirectly or that the fetal-placental unit increases the availability of nitric oxide in vivo. Therefore, despite what in vitro studies alone suggest, nitric oxide donor drugs may be very effective in suppressing either term or preterm labor.