The L1 metallo--lactamase from Stenotrophomonas maltophilia is unique amongst this class of enzymes in being tetrameric. Previous work predicted that the two regions of important inter-subunit interaction were the residue Met140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in -lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 -lactamase with greatly altered substrate specificity profile. A 20 amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form, but demonstrated greatly reduced rates of -lactam hydrolysis, and different substrate profiles compared to that of the parent enzyme. Specific site-directed mutations of individual N-terminus residues were made (Y11S, W17S, and a double mutant L5,8A). All N-terminal mutant enzymes were tetramers. and all showed higher Km values for ampicillin and nitrocefin, hydrolysed ceftazidime poorly, and hydrolysed imipenem more efficiently than ampicillin, in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably due to an increased rate of breakdown of the intermediate species due to a lack of stabilising forces. Km values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity which is not present in either the monomer or N-terminal deletion enzymes suggesting that the cooperative effect is mediated via N-terminal inter-subunit interactions. These data indicate that while the N-terminus of L1 is not essential for -lactam hydrolysis, it is clearly important to its activity and its substrate specificity.