The synthesis of the bacterial cell wall requires enzymes which are localized both in the cytoplasm and in the periplasm. Penicillin-binding proteins (PBPs) catalyze the last, crucial steps in peptidoglycan biosynthesis and several of them are essential for bacterial survival. High-molecular-mass PBPs can be bifunctional (class A) or monofunctional (class B) and to date no structural information on any class A PBP is available. To initiate the determination of the three-dimensional structure of a class A PBP, crystals of the transpeptidase domain of PBP1a from Streptococcus pneumoniae were prepared by limited proteolysis of the full-length molecule and purification by anion-exchange chromatography and gel filtration. The samples crystallize in space group C222(1), contain one molecule per asymmetric unit and diffract X-rays to 2.7 A. Selenomethionine-labelled crystals have been prepared and structure solution is under way.