Mechanisms and products of azo coupling in histochemical protease procedures based on primary aromatic amines as unspecific moieties. It is presumed that the azo dyes generated by histochemical protease reactions are formed by substitution of a reactive aromatic carbon. They are referred to as dyes of the C-azo series. To confirm this assumption, the absorption spectra between 330 and 630 nm of azo dyes resulting from coupling between various aromatic amines of the aniline and naphthylamine series and the diazonium salts Fast Blue B and Fast Garnet GBC were studied in test tube experiments. Some of the amines were blocked by methylation to prevent coupling either at the amino group (N-methylated) or at the aromatic nucleus (C-methylated). Coupling was performed in buffered aqueous solutions of the diazonium salts. For analysis the azo dyes were dissolved in dimethylformamide. For acid rearrangement these solutions were acidified and incubated at elevated temperatures. After detection of dipeptidyl peptidase IV in tissue sections using Gly-Pro-4-methoxy-2-naphthylamine as substrate, the resulting dye was extracted and compared with the test tube compounds.