The aim of the present study was to investigate the relationship between the expression of iNOS, COX-2 and VEGF mRNA levels and malignancy degree in canine malignant mammary tumours. Thirty-five bitches presented with the complaint of mammary masses, aged 6-10 years and representing different breeds, were used. The expressions of iNOS, COX-2 and VEGF mRNA levels were significantly higher in both benign and malignant tumours than in the adjacent non-neoplastic mammary glands (P < 0.05). The iNOS, COX-2 and VEGF mRNA expression levels of grade 2 tumours were higher than those of grade 1 tumours; however, the highest expression levels were detected in grade 3 tumours. Thus, increased iNOS, COX-2 and VEGF gene mRNA levels were found to be related with the histological grade of malignancy in dogs with mammary tumours.