Breast cancer is the most frequent spontaneous malignancy diagnosed in women in the western world, although no specific etiological agent(s) or the mechanism responsible for the initiation of the disease has been identified as yet. Enkephalins (Leu⁵-enkephalin and Met⁵-enkephalin) (ENK) act in the breast in different ways such as modulating esteroid receptors and proteases secretion. ENK are hydrolyzed by specific enzymes, leading to their inactivation, such as the enkephalin-degrading tyrosyl aminopeptidase (EDA). Breast tumours induced in rats by administration of N-methyl-nitrosourea (NMU) constitute a useful tool for dissecting the multistep process of carcinogenesis, which involves initiation, promotion and progression. The aim of the present work was to analyse EDA activity (E.C: 3.4.11.-) in serum of rats with mammary tumours induced by NMU, to evaluate the potential value of this activity as a biological marker of the carcinogenesis process, and the putative role of ENK in the promotion and progression of the disease. Tumours were induced by intraperitoneal injection of three doses of NMU at 50, 80 and 110 days after birth. Serum EDA was measured fluorimetrically using tyrosyl-,-naphthylamide as substrate. The increase found in EDA activity suggests the existence of decreased serum circulating levels of ENK in rat with mammary tumours induced by NMU. Although the exact role of ENK in breast cancer initiation, promotion and/or progression remains unknown, our results suggest that changes in EDA activity might play an important role in the origin and evolution of breast cancer.