The aim of this study was to assess a physiologically based modeling approach for predicting drug metabolism, tissue distribution, and bioavailability in rat for a structurally diverse set of neutral and moderate-to-strong basic compounds (n = 50). Hepatic blood clearance (CL(h)) was projected using microsomal data and shown to be well predicted, irrespective of the type of hepatic extraction model (80% within 2-fold). Best predictions of CL(h) were obtained disregarding both plasma and microsomal protein binding, whereas strong bias was seen using either blood binding only or both plasma and microsomal protein binding. Two mechanistic tissue composition-based equations were evaluated for predicting volume of distribution (V(dss)) and tissue-to-plasma partitioning (P(tp)). A first approach, which accounted for ionic interactions with acidic phospholipids, resulted in accurate predictions of V(dss) (80% within 2-fold). In contrast, a second approach, which disregarded ionic interactions, was a poor predictor of V(dss) (60% within 2-fold). The first approach also yielded accurate predictions of P(tp) in muscle, heart, and kidney (80% within 3-fold), whereas in lung, liver, and brain, predictions ranged from 47% to 62% within 3-fold. Using the second approach, P(tp) prediction accuracy in muscle, heart, and kidney was on average 70% within 3-fold, and ranged from 24% to 54% in all other tissues. Combining all methods for predicting V(dss) and CL(h) resulted in accurate predictions of the in vivo half-life (70% within 2-fold). Oral bioavailability was well predicted using CL(h) data and Gastroplus Software (80% within 2-fold). These results illustrate that physiologically based prediction tools can provide accurate predictions of rat pharmacokinetics.