The aim of this study was to develop a technique for separating monocytic cells in suspension from peripheral blood to measure the intracellular penetration of three fluoroquinolones (ofloxacin, ciprofloxacin and sparfloxacin). Mononucleated cells were isolated from the blood on a density gradient with lymphoprep and purified by a specific technique of adhesion and disadhesion on fibronectin. The monocytes were obtained in suspension with 76.8% purity and 97.9% viability. This was a convenient form for measurement of intracellular accumulation by use of the velocity-centrifugation technique. Intra-monocytic penetration of ciprofloxacin, ofloxacin and sparfloxacin was measured at equilibrium after 30-min incubation in the presence of 16 microg mL(-1) antibiotic. The results revealed low intra-monocytic accumulation of ciprofloxacin (intracellular-extracellular = 1.76) and ofloxacin (intracellular-extracellular = 1.42). The penetration of sparfloxacin was significantly higher (intracellular-extracellular = 2.4). This study confirms the important differences between human immunocompetent cells in terms of their ability to concentrate quinolones. It also underlines the importance of monocyte-macrophage cellular differentiation as a determinant of antibiotic penetration.