The absorption rates of 4-bromophenol, 2,4-dibromophenol, 3,4,5-trichloroguaiacol, and tetrachloroveratrol across fish gill epithelium have been measured in a perfused gill preparation from rainbow trout (Oncorhynchus mykiss). In additional experiments the absorption rate of tetrachloroveratrol was measured in free swimming fish. The absorption rates were perfusion limited in gills perfused with a saline containing 2% polyvinylpyrrolidone MW 40,000 (PVP-40) as a plasma protein substitute. Replacement of PVP-40 with 2% bovine serum albumin increased the absorption rates 5-10 times, and in this case rates were not perfusion limited. Furthermore, they were not ventilation limited at ventilatory water flow rates between 0.45 and 1.5 liter/(min 100 g). Our observations suggest that the absorption rates in the presence of albumin are limited by diffusion in the epithelial cell layers. The rates measured in the perfused gills are in close agreement with those measured in vivo. We conclude that the preparation is well suited to investigate the mechanisms behind the absorption of hydrophobic compounds across fish gills and how the absorption rate is related to the physicochemical properties of the compounds, as well as to environmental factors.