This study was performed to evaluate the distribution and depletion of sulfathiazole in different beehive matrices: honey, honeybees, "pre-existing" honeycomb, "new" honeycomb, and capping wax. Sulfathiazole was dissolved in sugar syrup or directly powdered on the combs, the matrices were sampled at different time points, and sulfathiazole residues were quantified by high-performance liquid chromatography with fluorescence detection. In honey, the higher concentration of sulfathiazole (180 mg kg(-1)) occurred 2 weeks after the last treatment in syrup. In beeswax, drug concentration was higher than in honey, particularly with powder administration, with a maximum level (340 mg kg(-1)) 3 days following the last treatment. The strongest contamination in honeybees (28 mg kg(-1)) was achieved with sulfathiazole administered in powder 3 days after the second treatment. The high persistence of sulfathiazole in the different beehive matrices suggests that it could be a reliable marker of previous treatments performed by beekeepers.