Information on the prevalence of subclinical endometritis and its mechanism in repeat breeding cows is very limited. The aims of this study were: a) to evaluate the incidence of this disorder with cytobrush cytology b) to analyze mRNA expression of tumor necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) in endometrial biopsy samples collected from repeat breeding cows with and without subclinical endometritis. Two experiments were carried out. In experiment 1,112 (12.4%) repeat breeding cows (inseminated at least 3 times and not pregnant) were selected out of 902 cows from 8 dairy herds. Cytobrush cytology was performed on these cows, using the threshold of 10% PMNs in uterine smears. The results showed that 45 out of the 112 cows (40.2%) were diagnosed as having subclinical endometritis. In experiment 2, uterine biopsy samples were taken from repeat breeding cows with subclinical endometritis (n = 10) and without this disorder (n = 10). Using reverse transcription-PCR, the mRNA expression of TNFalpha and iNOS was determined. A statistically significant increase in expression of both substances was measured in the group of cows with subclinical endometritis (p <0.05). These results provide evidence for a high prevalence of subclinical endometritis in repeat breeding cows as well as the involvement of TNFalpha and iNOS pathways in the regulation of this pathological condition.