Our objective was to examine the influence of ritonavir on P-glycoprotein (P-gp) activity in humans by characterizing the effect of ritonavir on the pharmacokinetics of the P-gp substrate digoxin in individuals with known MDR1 genotypes. Healthy volunteers received a single dose of digoxin 0.4 mg orally before and after 14 days of ritonavir 200 mg twice daily. After each digoxin dose blood and urine were collected over 72 hours and analyzed for digoxin. Digoxin pharmacokinetic parameter values were determined using noncompartmental methods. MDR1 genotypes at positions 3435 and 2677 in exons 26 and 21, respectively, were determined using PCR-RFLP analysis. Ritonavir increased the digoxin AUC0–72 from 26.20 ± 8.67 to 31.96 ± 11.24 ng · h/mL (P = 0.03) and the AUC0–8 from 6.25 ± 1.8 to 8.04 ± 2.22 ng · h/mL (P = 0.02) in 12 subjects. Digoxin oral clearance decreased from 149 ± 101 mL/h · kg−1 to 105 ± 57 mL/h · kg−1 (P = 0.04). Other digoxin pharmacokinetic parameter values, including renal clearance, were unaffected by ritonavir. Overall, 75% (9/12) of subjects had higher concentrations of digoxin after ritonavir administration. The majority of subjects were heterozygous at position 3435 (C/T) (6 subjects) and position 2677 (G/T,A) (7 subjects); although data are limited, the effect of ritonavir on digoxin pharmacokinetics appears to occur across all tested MDR1 genotypes. Concomitant low-dose ritonavir reduced the nonrenal clearance of digoxin, thereby increasing its systemic availability. The most likely mechanism for this interaction is ritonavir-associated inhibition of P-gp. Thus, ritonavir can alter the pharmacokinetics of coadministered medications that are P-gp substrates.