In the development of chemical cancer risk assessment methods, improvements in the ability to extrapolate across species depend not only on the availability of new data, but also on the understanding of species-specific biology of chemical handling. Thus, the need exists for quality comparisons of chemical behaviors between surrogate and target species, both toxicokinetically and dynamically. To reach these objectives, studies were performed to investigate the disposition of TCA in various tissues of rats exposed to TCE, characterize in vitro binding of TCA to plasma in human and rodents, and implement the binding behaviors into PBPK models for TCA tissue dosimetry in mice, rats, and humans following TCE exposure. In rats orally dosed with TCE (50 to 1000 mg/kg), the behaviors of TCA Cmax and AUCs were similar to those seen in mice at similar doses. Nonlinear dose-dependent increases in both dose metrics were seen in blood and tissues of the rat. At all doses, the mouse blood and tissue levels were higher than those of rats, presumably due to the differences in metabolic production of TCA. Such species differences in blood and tissue kinetics may have a significant impact on differences in susceptibility to TCE-induced liver cancer. Equilibrium dialysis was used to measure in vitro binding of TCA to plasma of humans, rats and mice. A model that provided for a single, saturable binding processes best fit the data. The greater plasma protein binding of TCA found in humans would be expected to increase the residence time of the compound in the bloodstream and reduce the proportion of TCA that is available for uptake by the liver and other tissues. A PBPK model for TCE in mice, rats, and humans that incorporated species-specific TCA plasma binding behavior was developed using the in vitro plasma binding data. Plasma binding had a dramatic, species-specific effect on the total and free fractions of TCA. The resulting extrapolated liver cancer risks suggested that the use of free blood TCA AUC rather than total blood TCA AUC produced a risk value better reflective of the actual target tissue exposure.