The sequential injection (SIA) technique was applied in pharmacokinetic studies of the transporter-mediated passage of a model substrate, rhodamine 123 (Rho123), through the dually perfused rat term placenta. The method described was used for real-time monitoring of Rho123 concentrations in both the maternal and fetal compartments. Determination was processed by fluorescence detection (lambda(ex)=480 nm, lambda(em)580 nm); calibration curve was linear over the range 0.01-50 mumoll(-1) (r=0.99965), detection limit was 10 nmoll(-1) (3sigma) and RSD2% (10 readings). Transport of Rho123 was scanned under various conditions (ATP-synthesis inhibition) and several inhibitors of P-glycoprotein transporter were tested (e.g. quinidine). The advantages of the modern SIA method-an automated analytical tool providing both fast and precise analysis-were successfully demonstrated for examination of transport profiles to investigate the effect of P-glycoprotein on the placental transfer of Rho123.