The impact of P-glycoprotein (P-gp) blockade on the intravenous (i.v.) pharmacokinetics of rhodamine-123 (Rho-123), and the subsequent effect on its disposition in ocular and nonocular tissues, was studied by using rabbits. Three (3) control rabbits received only an i.v. bolus dose of Rho-123 (1.52 mg/kg). Three (3) blocker-pretreated rabbits received an i.v. dose of GF120918 (3.5 mg/kg) 30 min before the i.v. bolus of Rho-123. The plasma concentration of Rho-123 at different time points was subjected to a pharmacokinetic compartmental analysis, using WinNonlin (Scientific Consultants, Lexington, KY). For tissue-distribution study, a drug treatment similar to the i.v. kinetic study was followed by having 5 rabbits in each group. The animals were sacrificed at 30 min with an excess of anesthesia. Plasma and tissues samples were analyzed by using a validated high-performance liquid chromatographic IV method with a fluorescent detector. The method validated was sensitive enough to estimate Rho-123 up to 1.94 ng/mL in plasma. I.v. Rho-123 data fitted well into the three-compartment model, and P-gp blocker treatment changed it into a two-compartment model. The P-gp blockade significantly increased the mean tissue concentrations in the lungs and spleen, whereas the rise in mean tissue levels in the heart, liver, and kidney and in all ocular tissues were found to be statistically insignificant. Increasing the ocular concentration of systemically given drugs may not be possible with the degree of P-gp blockade achieved when using GF120918 at the studied concentration after an i.v. administration.