Membrane-bound penicillin-binding proteins of an Escherichia coli carrying an R factor which mediated the resistance to penicillins were examined by slab gel electrophoresis and fluorography using beta-lactamase inhibitors such as methicillin, clavulanic acid and MC-696-SY2-A, and by affinity chomatography. By fluorography, it appeared that the penicillin-binding proteins of the strain carrying the R factor could not be distinguished from those of the parent strain. In both strains, methicillin had a preferential affinity for penicillin-binding proteins 2 and 3, clavulanic acid for 2 and 4, and MC-696-SY2-A for 1A at the concentration which was needed to inhibit about 75 approximately 80% of beta-lactamase activity of the membrane fraction from a strain carrying an R factor. This with other facts indicates that MC-696-SY2-A has a unique character in the binding to penicillin-binding proteins. By affinity chromatography using cephalexin-CH-Sepharose 4B column, two major cephalexin-binding proteins were detected. Their molecular weights were found to be 110,000 and 32,000, respectively. These two proteins correpsonded to penicillin-binding proteins 1 and 5/6. From these results it was suggested that the R factor had no influence on the penicillin-binding proteins in the E. coli strain examined.