BETA-Lactamases (EC. 188.8.131.52) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of beta-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate beta-lactamase in mutants previously reported to lack the enzyme. R 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, R1 and RGN14, with identical isoelectric focusing patterns have the same biochemical properties. Chromosomal and R-factor-mediated beta-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 beta-lactamases carried by R factors.