This study determined the unbound fraction of the peripheral α2 -adrenoceptor antagonist MK-467 alone and combined with medetomidine. MK-467 (0.1, 1 and 10 μm) was incubated in canine plasma with and without medetomidine (molar ratio 20:1), with human serum albumin (HSA) and with α1-acid glycoprotein (AGP). Rapid equilibrium dialysis was used for the measurement of protein binding. All samples were analysed by liquid chromatography and tandem mass spectrometry to obtain the unbound fraction (fu ) of MK-467. Unbound fractions (fu ) of MK-467 in canine plasma (mean ± standard deviation) were 27.6 ± 3.5%, 26.6 ± 0.9% and 42.4 ± 1.2% at 0.1, 1.0 and 10 μm concentrations, respectively. In the presence of medetomidine, fu were 27.5 ± 0.4%, 26.6 ± 0.9% and 41.0 ± 2.4%. The fu of MK-467 in HSA were 50.1 ± 2.5% at 0.1 μm, 49.4 ± 1.2% at 1.0 μm and 56.7 ± 0.5% at 10 μm. fu of MK-467 in AGP was 56.3 ± 3.7% at 0.1 μm, 54.6 ± 5.6% at 1.0 μm and 65.3 ± 0.4% at 10 μm. Protein binding of MK-467 was approximately 70% between 0.1 and 1.0 μm. Medetomidine had no apparent effect on the protein binding of MK-467.