Due to ubiquitous use of organophosphate esters (OPEs) as flame retardants and plasticizers, they have been found at high concentrations in a variety of environmental media while having low or non-detectable levels in biota, including fish. The exposure and fate of OPEs in fish depends on understanding the toxicokinetics, though studies on OPE metabolism are lacking. A model in vitro microsomal-based metabolism assay for rainbow trout (Oncorhynchus mykiss) was utilized to investigate OPE metabolism. Metabolic depletion was only noted for 2 alkyl OPEs and formation of corresponding diester metabolites was low, indicating other unidentified metabolites were formed. Comparison with avian and mammalian models showed fish generally had slower and less complete metabolism, illustrating species-specific differences in biotransformation. Structure also influenced OPE metabolism in that bulky, aryl OPEs were not metabolized. These results provide important information of toxicokinetic processes that can affect the exposure and fate of OPEs in rainbow trout.