ABSTRACT Beta-lactam antibiotics are often the treatment of choice for serious bacterial infections. In a previous screen for novel genetic mediators affecting beta-lactam susceptibility, we discovered that deletion of ydgH , a conserved gene of unknown function, leads to increased resistance to beta-lactams, as well as increased susceptibility to detergent compounds. Here, we further characterize YdgH in Serratia marcescens, Enterobacter cloacae , and Escherichia coli using a combination of biochemical and cell biological approaches. We find that YdgH fractionates with periplasmic proteins, and this periplasmic localization is necessary for its function. Using purified recombinant protein, we demonstrate that YdgH is a relatively compact, globular monomer. The YdgH polypeptide contains three tandem DUF1471 domains. In a Δ ydgH background, overexpression of polypeptides containing both the second and the third, but not the first DUF1471 domain, is necessary to rescue the deletion phenotype. To determine how YdgH function influences beta-lactam and detergent susceptibility, we tested several targeted hypotheses. We found that deletion of ydgH neither affects ompC or ompF transcript levels , nor does it alter the processing of lipopolysaccharide, nor does it activate the sigma E regulon alone or in combination with mutations in other periplasmic proteins. Finally, we delineate the results of a genetic screen for spontaneous mutants that complement the detergent susceptibility phenotype, the results of which may fuel the further studies that are necessary to determine the precise role YdgH plays in bacterial physiology. IMPORTANCE Beta-lactams such as penicillins and cephalosporins are the most commonly prescribed antibiotics for serious bacterial infections. Increasing antibiotic resistance threatens their effectiveness. We previously identified the uncharacterized gene ydgH as a modifier of beta-lactam susceptibility in Gram-negative bacteria. To begin to understand the specific role of YdgH, in this study, we perform initial characterizations of this protein. We also test hypotheses as to how the function of YdgH contributes to beta-lactam physiology.