Abstract Firemaster® BZ-54 is a flame retardant additive and consists of a brominated benzoate (2-ethylhexyl 2,3,4,5-tetrabromobenzoate; TBB) and a brominated phthalate (bis (2-ethylhexyl) 2,3,4,5-tetrabromophthalate; TBPH). Previous research has shown that fathead minnows exposed in vivo to Firemaster® BZ-54 accumulate TBB and TBPH. This study examined the in vitro biotransformation potential of TBB and TBPH in hepatic subcellular fractions (i.e., S9, microsomes and cytosol) in the fathead minnow, common carp, mouse and snapping turtle. Metabolism was evaluated by measuring the loss of the parent TBB or TBPH and identifying potential metabolites in the sample extracts. Metabolic loss of TBPH was measured for all species, while TBB loss was observed for all species except for the snapping turtle. Several metabolites were observed in all of the incubations except for snapping turtle. Metabolites observed appeared to be derived from TBB, given their structures and lack of appearance in the snapping turtle incubations. One of these metabolites, 2,3,4,5-tetrabromomethylbenzoate has been identified for the first time in a biological system. When metabolized, TBB and TBPH loss was found in each subcellular fraction suggesting that the enzyme(s) involved are present in both soluble and membrane-bound forms. It can be concluded that a broad range of species are capable of metabolizing TBB and TBPH to various metabolites and further research should be carried out to ascertain the specific products formed from metabolism of TBB and TBPH.