The expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS, respectively) was examined in the endometrium of cyclic and pregnant mares by real-time polymerase chain reaction and immunohistology. The concentration of eNOS mRNA varied throughout the oestrous cycle, with significantly higher transcripts on Day 5 of the oestrous cycle (P < 0.05), whereas iNOS transcription did not change significantly over time (P > 0.05). In early pregnant mares both eNOS and iNOS mRNA increased between Days 12 and 15 (P < 0.05). In cyclic mares, eNOS protein was detected immunocytochemically in endometrial epithelia, the basement membrane, the endothelial layer and smooth muscle cells of the vasculature. Using immunocytochemical methods, iNOS protein was undetectable in the endometrium of cyclic mares but could be demonstrated in pregnant mares. Endometrial epithelia of pregnant mares were immunopositive for both proteins with a more intense labelling for iNOS. Thus, the present study describes for the first time the modulation and spatial distribution of eNOS and iNOS expression during the oestrous cycle and early pregnancy, suggesting that ovarian steroids are differently involved in the regulation of each NOS. Localisation of eNOS protein in endometrial epithelia and various vascular components indicates that this isoform may be involved in the regulation of endometrial cyclicity. The presence and increase of both forms of NOS during early gestation suggest a role for them in the control of endometrial vascular bed and glandular activity to provide a suitable microenvironment for successful pregnancy.