The objective was to investigate the interplay between transporter expression levels and substrate affinity in controlling the influence of aqueous boundary layer (ABL) resistance on transporter kinetics in an over-expression system. Taurocholate flux was measured across human apical sodium-dependent bile acid transporter (hASBT)-Madin-Darby canine kidney monolayers on different occasions and kinetic parameters estimated with and without considering ABL. In error-free simulation/regression studies, flux values were generated across a range of J(max), K-t, and substrate concentrations. Similar evaluation was performed for transport inhibition studies. Additionally, simulation/regression studies were performed, incorporating 15% random error to estimate the probability of successfully estimating K-t. Across different occasions, experimental J(max) and K-t estimates for taurocholate were strongly associated (p < 0.001; r(2) = 0.82) when ABL was not considered. Simulation/regression results indicate that not considering ABL caused this association, such that K-t estimates were highly positively biased at high hASBT expression. In reanalyzing taurocholate flux data using the ABL-present model, K-t was relatively constant across occasions (similar to 5 mu M) and not associated with J(max) (p = 0.24; r(2) = 0.13). Simulations suggest that J(max) and K-t collectively determined ABL influence, which is most prominent under conditions of low monolayer resistance. Additionally, not considering ABL lead to negatively biased K-i estimates, especially at high Jmax. Error-inclusive simulation/regression studies indicated that the probability of successfully estimating K-t depended on the contribution of ABL resistance to flux; when flux became increasingly ABL-limited, probability of success decreased. Results indicate that ABL resistance can bias K-t and K-i estimates from overexpression systems, where the extent of bias is determined by transporter expression level and substrate affinity.