The development of high level beta-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant S. pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime = 12 mg.L(-1)). Mutations T371A, which is adjacent to the catalytic nuclophile Ser370, and TSQF(574-577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with beta-lactams were probed in vitro, and their effect on resistance were measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared to that of PBP1a from the beta-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370.