S U M M A R YP-Lactamases (EC. 3 . 5 . 2 . 6 ) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of P-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate P-lactamase in mutants previously reported to lack the enzyme. R factor RTEM and RPI P-lactamases that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R I and RGNI1, with identical isoelectric focusing patterns, have the same biochemical properties. Chromosomal and R factor-mediated P-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing, R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of P-lactamases carried by R factors. I N T R O D U C T I O NIsoelectric focusing (Haglund, 1967) is a method of separation in which proteins align themselves as sharp bands at their isoelectric points (PI) in an electrophoretically-produced pH gradient. A high degree of resolution is obtained by the method, because focusing is caused by forces that act against diffusion and proteins are therefore concentrated during their separation. We have used the technique in the analytical mode for the separation of P-lactamases (EC. 3 . 5 . 2 . 6 ) on thin layers of polyacrylamide gel and have located the focused enzymes with a cephalosporin substrate which changes colour after degradation by P-lactamase. This technique of separation and specific staining of P-lactamases allows demonstration of low levels of activity and presents the different enzymes produced by various strains as patterns of bands that can easily be recognized and compared.The P-lactamases produced by Gram-negative bacteria are known to differ in their substrate specificities, inhibitor profiles, electrophoretic mobilities, biochemical properties and reactions with antisera ( Yamagishi et al. 1969). These enzymes have frequently been classified by the relative rates at which they hydrolyse penicillins and cephalosporins. Various methods have been used for estimating the activity of crude and pure enzyme preparations, and when a single strain produces two P-lactamases these have not always been separated before determining the substrate profile. Comparison of profiles determined in different laboratories may therefore be misleading. The use of isoelectric focusing of P-lactamases for direct comparison of different enzymes has been illustrated in this paper principally by reference to enzymes from Escherichia coli. This species was chosen because strains carrying R factors specifying well-characterized P-lactamases were available. METHODSStrains. These are listed in Table I. The clinical ...