This study was carried out to investigate the most important factor(s) governing the tissue distribution of grepafloxacin (GPFX), a fluoroquinolone antibiotic, in rats. The tissue-to-blood concentration ratio (K(p)) of GPFX at steady state during constant infusion was highest in the lung, followed by the pancreas, kidney, and spleen. After bolus injection, GPFX was efficiently taken up by most of the organs examined, the uptake clearance other than the lung being almost blood flow-limited. Approximately 10% of the intravenously injected dose was rapidly trapped by the lung, but GPFX distribution rapidly decreased within 30 s due to the washout by the plasma flow. Thus, the higher distribution of GPFX to the lung compared with the other organs cannot be accounted for by a difference in its uptake or efflux. Subcellular fractionation after the infusion indicated that GPFX is primarily distributed to the organelle fractions in most organs, 60% of lung-associated GPFX being recovered in the nucleus and plasma membrane fraction. Such subcellular distribution in the lung was proportional to the phosphatidylserine (PhS) content of each fraction. The steady-state K(p) value in each tissue in vivo also correlated with the tissue content of PhS. GPFX preferentially binds to PhS, compared with other phospholipids, and this binding was inhibited by weakly basic drugs, such as quinidine, imipramine, and propranolol, that have also been reported to bind to PhS. The association of GPFX with PhS synthase transformants of Chinese hamster ovary (CHO-K1) cells depends on the PhS content of each cell line, this association being also inhibited by basic drugs. These results suggest that binding of GPFX to PhS is the major determinant of the high distribution of GPFX to the lung.