Thymidine kinase 1 (TK1) is a deoxyribonucleic acid (DNA) precursor enzyme and a proliferation biomarker used for prognosis and treatment monitoring of breast cancer in humans. The aim was to determine if serum thymidine kinase 1 (sTK1) activity and sTK1 protein levels in dogs with mammary tumors could be useful in veterinary medicine.,Serum samples from 20 healthy dogs and 27 dogs with mammary tumors were analyzed for sTK1 activity, using an [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for sTK1 protein levels by immune affinity/Western blot assay. The molecular forms of sTK1 in acute lymphocytic leukemia (ALL), canine mammary tumor (CMT), and healthy sera were determined by size exclusion chromatography. Mean sTK1 activities in CMT were 1.0 ± 0.36 pmol/min/mL, differing significantly from healthy dogs (mean ± SD = 0.73 ± 0.26 pmol/min/mL). Serum TK1 protein (26 kDa polypeptide) levels were also significantly higher in CMTs compared to healthy dogs (mean ± SD = 28.5 ± 11.4, and 8.5 ± 4 ng/mL, respectively). Cellular TK1 isolated from ALL tumor cells was predominantly a dimer, while the serum TK1 activity eluted as a high molecular weight (MW) oligomer. In analyses of CMT tissue extracts, TK1 activity eluted in two peaks, a minor peak with a high MW oligomer and a major tetramer peak. Western blot analysis of chromatographic fractions showed that cellular TK1 protein in both ALL and CMT dogs, and to some extent serum TK1 from ALL dogs, correlated with activity profiles, but a large fraction of inactive TK1 protein was detected in CMT.,Serum TK1 protein and activity levels were significantly higher in CMT than in healthy dogs. Size exclusion chromatography demonstrated major differences in the molecular forms of sTK1 in ALL, healthy, and CMT dogs, with a large fraction of inactive TK1 protein in CMT. Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay. This preliminary data supports that sTK1 protein assay is clinically useful. Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs.