Chromosomally encoded ss-lactamases from the Burkholderia cepacia complex species (formerly Pseudomonas cepacia) were characterized. Cloning and sequencing identified an Ambler class A ss-lactamase (PenB) from B. cenocepacia. It shares 82% amino acid identity with the PenA ss-lactamases previously identified from B. multivorans 249. Its expression was dependent upon a LysR-type regulatory protein. Its narrow-spectrum hydrolysis activity mostly included penicillins but also included expanded-spectrum cephalosporins and aztreonam at lower levels. In that study, Pen-like ss-lactamases (PenC, PenD, PenE, PenF) that shared 63 to 92% identity with PenB from B. cenocepacia were identified from other Burkholderia species. The corresponding ss-lactamase genes might be used as genetic tools for accurate Burkholderia species identification.