An evaluation was made of the role of the outer membrane of Haemophilus influenzae type b as a permeability barrier against beta-lactam antibiotics. Sonic extracts of H. influenzae containing beta-lactamase were assayed for the rates of hydrolysis of benzylpenicillin, ampicillin, cloxacillin, cephacetrile, cefazolin, cefamandole, cephalothin, cephaloridine, cephaloglycin, and cefaclor. Benzylpenicillin was hydrolyzed most rapidly, whereas cephacetrile, cephaloridine, and cephaloglycin were the poorest substrates for the beta-lactamase. The hydrolysis of these ten beta-lactams by intact cells was also determined; it was necessary to stabilize the cells with MgCl2 to prevent lysis and thereby to maintain the beta-lactamase in the periplasm. Calculations were made of the concentration of the antibiotics which had accumulated in the periplasm. The transmembrane permeability coefficient, C, was determined for the ten beta-lactam antibiotics. All of the compounds tested were able to diffuse across the outer membrane of H. influenzae type b very efficiently. The values of the permeability coefficient were compared with the partition coefficients of the antibiotics in a two-phase isobutanol/water mixture. For a ten-fold increase in hydrophobicity, there was a ten-fold decrease in the permeability coefficient. The outer membrane of haemophilus was not an effective barrier against the penetration of penicillins or cephalosporins. The activity of these compounds could be attributed either to their low hydrolysis by beta-lactamase or to the high affinity of binding to their sensitive targets.