2-Nitro-4-azidophenylsulphenyl-glucagon, a specific photoaffinity label for glucagon receptors, was synthesized and radioactively labelled with 125I. The radio-labelled peptide was purified from the reaction mixture by high-performance liquid chromatography in one step by isocratic elution from a C18 column with 20.4% n-propanol in 10 mM phosphate buffer (pH 2.5) as eluent. This glucagon derivative can be used to attach a label specifically to the glucagon receptor. The binding ability of the photoaffinity derivative was tested on isolated intact rat hepatocytes. Compared with a Kd of 3 nM for unmodified monoiodinated glucagon, the Kd value of the photoaffinity labelled monoiodinated glucagon tracer was 7 nM.