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The objective of this study was to investigate the relationships between uterine perfusion and estrogen, progesterone and the uterine nitric oxide synthase (NOS) system in five trotter mares during the estrous cycle. Color Doppler sonography for measurement of uterine blood flow and collection of blood for determination of plasma estrogen and progesterone concentrations were performed on days 0 (= ovulation), 1, 5, 11 and 15 and daily during estrus (days -1 to -4) of one estrous cycle; endometrial biopsy collection for mRNA expression analysis of NOS and estrogen receptors was performed on days 0, 1, 5, 11, 15 and -3. Blood flow in each uterine artery was assessed by calculating the mean time-averaged maximum velocity (TAMV) and the pulsatility index (PI). Plasma concentrations of estrogen and progesterone were determined using specific enzyme immunoassays. The mRNA expressions of endothelial NOS (eNOS), inducible NOS (iNOS) as well as estrogen receptors α (ERα) and β (ERβ) were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The TAMV and PI had a biphasic pattern during the estrous cycle (P<0.05), with maximum and minimum, respectively, values on days 5 and -4. Estrogen receptor mRNA concentrations increased significantly during days 15 (ERα) and -3 (ERβ). Transcript expression of eNOS, but not iNOS, had a biphasic pattern during the cycle (P<0.05) with maximum levels on days 5 and -3 and correlated positively with TAMV (r=0.81, P=0.05). We infer that the uterine NOS system, especially eNOS, plays an important role in the regulation of uterine blood flow during the estrous cycle in mares.