Azo compounds are extensively used for colouring food, drink, pharmaceuticals, cosmetics, textiles and printing inks. Publications in the literature have shown that azo dyes can pose threats to public health by metabolic and chemical oxidation and reduction reactions. In the present study, the in vivo and in vitro biotransformation of ethyl 4-[(2-hydroxy-1-naphthyl)azo]benzoate, an azo compound which is structurally similar to 1-phenylazo-2-naphthol was studied to investigate its in vivo and in vitro metabolic products. For the in vitro biotransformation study, rat liver microsomal preparations fortified with NADPH as a co-factor were used. Three unidentified metabolic products were observed. For the in vivo biotransformation study, a concentrated solution of this substrate was given orally to female rats. After the administration of substrate, blood samples of rats are taken at certain intervals. The blood plasma were obtained by centrifuging blood samples. The cold acetonitrile was added to plasma to precipitate plasma proteins and plasma was centrifuged. The supernatant was evaporated at room temparature. The residue was reconstituted with acetonitrile and examined by the HPLC. The unchanged substrate together with the corresponding reduction and acetylation products were detected in plasma. However, no initial hydrolysis occurred in the ester moiety.