Nitric oxide (NO) is a free radical produced by the action of nitric oxide synthases (NOS) and is known to be involved in the regulation of many reproductive events that occur in the oviducts. The oviducts are highly specialized organs that play crucial roles in reproduction by providing an optimal environment for the final maturation of gametes, fertilization and early embryo development. In this study, we analyzed the expression, the hormonal regulation, and the cellular distribution of neuronal, inducible and endothelial NOS in different bovine oviduct segments to better understand the roles played by these enzymes in oviductal functions in vivo. Quantitative RT-PCR analysis revealed that NOS isoforms are hormonally regulated and differentially expressed along the oviduct throughout the estrous cycle. All NOS were highly expressed around the time of estrous and immunohistochemistry studies determined that nNOS, iNOS and eNOS are differentially distributed in cells along the oviduct. Interestingly, our results showed that estradiol selectively up-regulates iNOS expression in the oviduct during the peri-ovulatory period corresponding to the window of ovulation, oocyte transport and fertilization. The resulting NO production by this high-output NOS may be of crucial importance for reproductive events that occur in the oviduct. This study provided the first demonstration that NO production is hormonally regulated in the mammalian oviducts in vivo. Our results suggest that nNOS, iNOS and eNOS contribute to oviductal functions in a timely and site-specific manner.