OBJECTIVE Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis and an important proliferation marker. We explored the association of preoperative serum TK1 activity with clinicopathological parameters and prognosis in terms of recurrence-free survival (RFS) in breast cancer (BC) patients. METHODS TK1 activity in serum of 120 healthy women and 161 BC patients was measured by quantitative ELISA. RESULTS Serum TK1 activity in BC patients was significantly higher than in healthy women (P < 0.0001). In BC patients elevated TK1 activity was significantly associated with advanced T stage (P = 0.015), higher grade (P = 0.013), presence of tumor necrosis (P = 0.006), vascular invasion (P = 0.002), and lack of estrogen receptor (ER) and progesterone receptor (PR) expression (P = 0.0004 and P = 0.003). Higher TK1 activity was found in patients with BRCA1/2 mutations compared to those without the mutation (P = 0.004). Multivariate Cox proportional hazards analyses demonstrated that TK1, adjusted for stage, grade, necrosis, ER and PR negativity was retained as an independent predictor of disease recurrence (Hazard Ratio = 3.9, 95%CI 1.3-11.6, P = 0.013). CONCLUSIONS Elevated serum TK1 is an important risk factor indicating a high proliferation potential of tumors at the time of excision. In multivariate analysis TK1 activity was found to be an independent prognostic factor for RFS.