An enzyme-linked immunosorbent assay was developed for the determination of canine β₂-microglobulin (β₂-m) in plasma and urine. The detectable sensitivity for pure canine β₂-m was 0.05 μg/l and the analytical range was 0.1 to 50 μg/l. The mean analytical recovery when pure canine β₂-m was added to normal plasma was 101.9%. The mean analytical recovery in the urine was 102.1%. The intra-day variation coefficient was 3.1% in plasma, 4.3% in serum and 1.9% in urine. No difference was found between the concentration of β₂-m in plasma and serum (n=17). The concentration of β₂-m in the plasma of normal dogs was 1.82 ± 0.57 mg/l (n=31). The mean excretion in 24 hr urine collected from normal dogs was 17.6 ± 9.2 μg/l, 0.22 ± 0.12 μg/kg of body weight or 14.2 ± 9.4 μg/g of urine creatinine. The β₂-m creatinine index of random urine samples was 23.5 ± 16.6 μg/g (n=26). There was a close correlation between the β₂-m creatinine index of 24 hr urine samples and that of random urine samples (r= 0.872).