As part of a larger clinical drug-drug interaction (DDI) study aimed at in vitro to in vivo prediction of HIV protease inhibitor metabolic and transporter-based DDIs, we measured the inductive (staggered administration) and inductive plus inhibitory (simultaneously administered) effect of multiple dose ritonavir (RTV), nelfinavir (NFV), or rifampin (RIF) on the pharmacokinetics of the P-glycoprotein probe, digoxin (DIG), when administered simultaneously or staggered with the protease inhibitors or RIF. In both cases, NFV did not significantly affect DIG disposition. RTV decreased DIG renal clearance (Cl(renal)) when administered simultaneously or staggered but significantly increased DIG area under the curve from time zero to 24 h (AUC(0-24 h)) only when administered simultaneously. RIF decreased DIG AUC(0-24 h) only when RIF and DIG administration was staggered. When RIF and DIG were administered simultaneously, DIG maximal observed plasma concentration and area under the curve from time zero to 4 h were significantly increased, and DIG Cl(renal) was decreased. An unexpected and potentially clinically significant DDI was observed between DIG and the CYP2B6 probe, bupropion, which decreased DIG AUC(0-24 h) 1.6-fold and increased Cl(renal) 1.8-fold. Because this was an unexpected DDI and our studies were not specifically designed to quantify this interaction, further studies are required to confirm the interaction and understand the mechanistic basis of the DDI. In summary, RTV or NFV do not induce P-glycoprotein activity measured with DIG, and RIF does so only under staggered administration.