Despite their clinical importance, the mechanisms by which β-lactams inhibit penicillin-binding-proteins (PBPs) are not fully defined. X-ray crystallographic and mass spectrometric data studies of a Pseudomonas aeruginosa PBP3 active site variant (Tyr409Ala) reveal evidence for the in vitro acylation of P. aeruginosa PBP3 by reaction of nitrocefin with both Ser294 and Ser349. The results reveal a potentially novel binding mode for β-lactams with PBPs and will inform the design of new PBP inhibitors.