The unidirectional influx of benzylpenicillin through the blood-brain barrier was examined with an in situ rat brain perfusion technique using purified [3H]benzylpenicillin. The major transport system of benzylpenicillin through the blood-brain barrier was via a saturable process with a one-half saturation concentration of approximately 8-30 microM. This transport system was significantly inhibited by probenecid (100 microM) and ceftriaxone (2 mM), indicating that the transport system may be shared by some organic anions including third generation cephalosporin antibiotics. These findings suggest that concomitant administration of beta-lactam antibiotics could produce a drug interaction to alter the drug penetration into the central nervous system.