Victoria Pure Blue BO is a dye that bears some therapeutic activity and that can be retrieved in effluent or may be used in aquaculture as a prohibited drug. In this study, the metabolism and tissue distribution during uptake and depuration of VPBO were investigated in order to propose a residue marker of illegal treatment in fish. The dye was administered to rainbow trout (oncorhynchus mykiss) for one day by water bath at a dose of 0.1 mg.L(-1). The concentrations of VPBO in all tissues increased rapidly during the treatment period, reaching a C(max) of 567 ± 301 μg.L(-1) in plasma and 1846 μg kg(-1) ±517 for liver after 2 h. After placing the rainbow trout in a clean water bath for a 64 day-period of depuration, the concentrations in the tissues and plasma decreased to reach comparable levels for muscle and for skin after 33 days. The concentrations measured were still above the LOQ at 2.26 ± 0.48 μg kg(-1) for muscle and 2.85 ± 1.99 μg kg(-1) for skin at the end of the depuration period. The results indicated the existence of 14 phase I metabolites and one glucuronide conjugated metabolite. Non-compartmental analysis was applied to assess the pharmacokinetic parameters. The half-life in edible muscle of the main metabolite detected, deethyl-leuco-VPBO, was found to be 22.5 days compared to a half-life of 19.7 days for the parent VPBO. This study provides new information to predict a VPBO drug treatment of aquacultured species via a proposed new residue marker.