1. The intestinal permeability and hepatic metabolism of the investigational cancer chemoprevention agent 4'-bromoflavone were investigated in vitro using human intestinal Caco-2 cell monolayers, human liver microsomes and human hepatocytes. Liquid chromatography-mass spectrometry and tandem mass spectrometry were used for quantitative analysis in support of the Caco-2 cell studies and for the characterization of metabolites of 4'-bromoflavone. 2. The Caco-2 cell model indicated that 4'-bromoflavone would be absorbed by the intestine at a moderate rate by means of direction-independent, passive diffusion. There was no indication of active transport or efflux. 3. Three monohydroxylated metabolites and one monohydroxylated, hydrated metabolite of 4'-bromoflavone were detected at relatively low levels in the human liver microsomal and hepatocyte incubations. The structures of these metabolites were confirmed by comparison with synthetic standards. Hydroxylation occurred on the A-ring of 4'-bromoflavone but not on the B-ring, probably due to deactivation of the B-ring by bromine. No phase II metabolites were detected following incubation of 4'-bromoflavone in these in vitro systems. 4. In conclusion, these studies predict that 4'-bromoflavone should show moderate oral bioavailability, and that it would probably be excreted as unchanged compound and monohydroxylated metabolites. The results might be helpful in the design of clinical trials and in the interpretation of pharmacokinetic studies of 4'-bromoflavone.