PBP2a works in a similar manner to other PBPs, but it binds β-lactams with very low affinity, meaning they do not compete efficiently with the natural substrate of the enzyme and will not inhibit cell wall biosynthesis.

[citation needed] These susceptibility data are given on a few medically significant bacteria: Like other beta-lactam antibiotics, methicillin acts by inhibiting the synthesis of bacterial cell walls.

It inhibits cross-linkage between the linear peptidoglycan polymer chains that make up a major component of the cell wall of gram-positive bacteria.

Methicillin and other β-lactam antibiotics are structural analogs of D-alanyl-alanine, and the transpeptidase enzymes that bind to them are sometimes called penicillin-binding proteins (PBPs).

[4] It was previously used to treat infections caused by susceptible gram-positive bacteria, in particular, penicillinase-producing organisms such as Staphylococcus aureus that would otherwise be resistant to most penicillins.