It was suggested previously that the primary structure of penicillin-binding protein 4 (PBP4) is new and unique among proteins that interact with penicillin. Our proposal that PBP4 carries an additional domain, located between the active-site fingerprints SXXK and SXN, was investigated by mutational deletion analysis. A clustered set of internal deletions was created in this region by exonuclease treatment of the dacB coding DNA, starting from two internal restriction sites. PBP4 mutants carrying internal deletions were selected by screening for immunoreactive forms of PBP4 with reduced molecular weight that were still active with respect to penicillin binding. DNA sequencing revealed 24 distinct PBP4 mutants with internal deletions ranging from 37 to 113 amino acids. The amino- and carboxy-terminal end points of the deletions were not randomly distributed but tended to cluster in certain areas. Overproduction of the individual mutated forms of PBP4 resulted in accumulation of the major portion of the proteins in the particulate cell fraction. The yield of soluble and active mutated forms of the protein was reduced from below 1% to 79% of the level obtained for the native protein. The deletions that were introduced had minor effects on the deacylation rate of bound benzylpenicillin. Two pairs of cysteine residues (Cys-139-Cys-153 and Cys-197-Cys-214) that are located in the deletable region may form disulfide bridges.