A four-step strategy is proposed for determining appropriate experimental designs for investigating the pharmacokinetics of drugs characterized by complex compartmental models and this strategy has been applied to the pharmacokinetics of enterohepatic circulation (EHC). The four steps are (1) to establish an appropriate pharmacokinetic model, (2) to complete an identifiability analysis for the model to determine the route(s) of administration and sampling compartment(s) that are theoretically adequate for the quantitation of model parameters, (3) to carry out nonlinear least-squares fitting for the proposed number and timing of simulated error-free data points, and (4) to complete nonlinear least-squares fits of the model to data obtained by adding random error to the simulated data in step 3. The four-compartment model chosen for EHC of unchanged drug contained central, peripheral, gallbladder, and intestinal compartments and an intermittent gallbladder emptying rate constant. Identifiability analysis demonstrated that three alternative experimental designs for route(s) of administration and sampling compartment(s) are adequate for quantitating all model parameters, when the gallbladder emptying rate constant as a function of time is known (using controlled emptying from an engineered gallbladder in an animal model or quantitation in humans or animals using imaging techniques). Parameter estimates from fitting error-free data matched closely with the known values for all three experimental designs, indicating an adequate number and appropriate timing of data points. Results from fitting simulated data containing +/- 10% random error indicated unacceptable coefficients of variation and a nonrandom pattern in residual plots for one of the experimental designs.(ABSTRACT TRUNCATED AT 250 WORDS)