The New Delhi metallo-b-lactamase (NDM-1) mediates resistance to b-lactam antibiotics. NDM-1 was created by a fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-b -lactamase. We show that NDM-1 has an atypical signal peptide and is ineffficiently secreted. Using mass spectrometry we show that NDM-1 is cleaved by E. coli signal peptidase I between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. Two new blaNDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A). We show that both the P9R and P28A substitutions improve secretion compared to NDM-1. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. NDM-1(P9R) and NDM-2 exhibit improved secretion, increased resistance to some antibiotics and expression of NDM-2 improves the fitness of E. coli compared to NDM-1, in the absence of antibiotic selection. Further optimization of the secretion efficiency of this metallo-b-lactamase may give rise to new alleles with increased resistance and pathogens with increased fitness.