The first step in the biosynthesis of the medicinally important carbapenem family of ß-lactam antibiotics is catalysed by carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily. CarB catalyses formation of (2S,5S)-carboxymethylproline [(2S,5S)-t-CMP] from malonyl CoA and L-glutamate semialdehyde. In addition to using a cosubstrate, CarB catalyses C-C and C-N bond formation processes as well as an acyl-coenzyme A hydrolysis reaction. We describe the crystal structure of CarB in the presence and absence of acetyl CoA at 2.24 Å and 3.15 Å resolution, respectively. The structures reveal that CarB contains a conserved ‘oxy-anion’ hole probably required for decarboxylation of malonyl CoA and stabilisation of the resultant enolate. Comparison of the structures reveals that conformational changes (involving His229) in the cavity predicted to bind L-glutamate semialdehyde occur on (co)substrate binding. Mechanisms for the formation of the carboxymethylproline ring are discussed in the light of the structures and the accompanying studies using isotopically labelled substrates; cyclisation via 1,4-addition is consistent with the observed labelling results (providing that hydrogen exchange at the C-6 position of carboxymethylproline does not occur). The side chain of Glu131 appears to be positioned to be involved in hydrolysis of the carboxymethylproline CoA ester intermediate. Labelling experiments ruled out the possibility that hydrolysis proceeds via an anhydride in which water attacks a carbonyl derived from Glu131, as proposed for 3-hydroxyisobutyryl CoA hydrolase. The structural work will aid in mutagenesis studies directed at altering the selectivity of CarB to provide intermediates for the production of clinically useful carbapenems.