The cephalosporins are a family of semisynthetic antibiotics, some of which have structural features associated with substrates for the multidrug transporter, P-glycoprotein. The activity of a series of six cephalosporins in reversing multidrug resistance (MDR) was examined in MDR variants (Dx5 cells) of the human sarcoma line MES-SA. Dx5 cells express high levels of the mdr1 gene product P-glycoprotein and are 25- to 30-fold resistant to doxorubicin (DOX), etoposide (VP-16), and vinblastine (VBL). Cytotoxicity was measured by the MTT assay. Cefoperazone (1.0 mM) was the most effective modulator of MDR, lowering the IC50 for VP-16 by 29-fold (29x), for VBL by 16x, and for DOX by 14x. Ceftriaxone at 1.0 mM produced 10x modulation of VP-16 cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of resistance was concentration dependent, decreasing to 4x and 5x, respectively, for DOX with 0.25 mM cefoperazone and ceftriaxone. No modulation of cytotoxicity was observed in the parental MES-SA cells, which do not express mdr1. Cefazolin, cefotetan, cephradine, and ceftazidime were ineffective, producing less than 5x modulation of DOX at 1.0 mM. Among these cephalosporins, cefoperazone and ceftriaxone were the most highly protein bound in the media (30 and 52%), and the most lipid soluble, with octanol/water partitioning coefficients of -0.49 and -0.60. Varying the serum concentration in medium from 5 to 50% had less than a two-fold effect on the modulation of MDR by ceftriaxone. The ability to reverse MDR among these agents is associated with lipid solubility, high protein binding, a polycyclic planar geometry, and the presence of the piperazine group in cefoperazone. These data and the potential for achieving high tissue concentrations indicate that cefoperazone merits further study as a modulator of MDR.