Abstract The backbone dynamics for the 29.5 kDa class A β-lactamase PSE-4 is presented. This solution NMR study was performed using multiple field 15N spin relaxation and amide exchange data in the EX2 regime. Analysis was carried out with the relax program and includes the Lipari-Szabo model-free approach. Showing similarity to the homologous enzyme TEM-1, PSE-4 is very rigid on the ps-ns timescale, although slower μs-ms motions are present for several residues; this is especially true near the active site. However, significant dynamics differences exist between the two homologs for several important residues. Moreover, our data support the presence of a motion of the Ω loop first detected using molecular dynamics simulations on TEM-1. Thus, class A β-lactamases appear to be a class of highly ordered proteins on the ps-ns timescale despite their efficient catalytic activity and high plasticity toward several different β-lactam antibiotics. Most importantly, catalytically relevant μs-ms motions are present in the active site, suggesting an important role in catalysis.