Resistance of gram-negative bacteria to beta-lactam antibiotics is based mainly on two mechanisms: hydrolysis by beta-lactamases and exclusion of the antibiotics from their target sites in the inner membrane. This article describes the use of Pseudomonas aeruginosa K 799/WT and a mutant of this strain (K 799/61) to assess the role of the outer membrane as a permeability barrier to penicillins and cephalosporins. The data confirm the importance of good penetration for a beta-lactam to be active against Pseudomonas. The second part illustrates the interplay of beta-lactamases and the outer membrane in the resistance of Escherichia coli to beta-lactams. A method to determine membrane permeability parameters parameters is given. The results support the idea that only a combined consideration of inactivating enzymes and penetration barriers can lead to a better understanding of the efficiency of the defence mechanisms which gram-negative bacteria can invoke against beta-lactam antibiotics.