Nitric oxide (NO) production in the rat placenta was monitored and quantified by electron paramagnetic resonance (EPR) spectroscopy with hemoglobin and an Fe-N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trapping reagents. Expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative RT-PCR analysis. The EPR spectrum of the placenta with hemoglobin trapping showed a three-line hyperfine structure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOS inhibitor L-NAME was administered to pregnant rats. Therefore, the specific signal was definitely identified as being derived from endogenous NO spin-trapped by hemoglobin, and the EPR spectrum showed that the NO adduct existed as a pentacoordinate -NO heme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from an NO-Fe-DTCS complex. The height of the triplet signal did not vary significantly with gestational stage during the last few days of gestation. At the gestational stages examined, the level of NOS II mRNA expression was significantly higher than that of NOS III mRNA. NOS II expression in term (day 21.5) placenta was significantly increased compared with that in preterm (day 19.5) placenta (P < 0.01, n = 4 or 5). These results suggest that NOS II is the predominant producer of NO in the placenta and that NOS II-generated NO plays significant roles in the maintenance of placental functions immediately before birth.