A series of mutants defective in penicillin-binding components (or protein) (PBCs) was isolated from a collection of thermosensitive mutants of Escherichia coli. The mutants included mutations for each PBC (1 through 4) and a mutation in the activity of D-alanine carboxypeptidase 1a (PBC-5/6). PBC-1 was resolved into two components, PBC-1a and PBC-1b, which were the products of different genes referred to as ponA and ponB, respectively. No mutation examined in ponA-- or ponB--, by itself, was associated with a thermosensitive defect in growth. The mutants having ponB-- were hypersensitive to cephalosporins, which showed unusually high affinity to PBC-1a. Murein synthetic activity decreased markedly in cell-free preparations from the ponB-- mutant. The double mutation ponA-- ponB-- was found to be lethal. A mutant having ponAts and ponB-- revealed thermosensitivity of growth and lysed at the restrictive temperature. It is concluded that PBC-1a and PBC-1b could share a biochemical reaction necessary for cell elongation so that the function of either one may bypass the loss of the function of the other. With the series of mutants, the chromosomal locus of the gene for each PBC was mapped: ponA (PBC-1a) 73.5 min, ponB (PBC-1b) 3.3 min, rodA (PBC-2) 14.4 min, ftsI (PBC-3) 1.8 min, dacB (PBC-4) 68 min, and dacA (PBC-5/6) 13.7 min.