The gene for proline iminopeptidase from Lactobacillus delbrueckii subsp. lactis DSM 7290 coding for an enzyme that hydrolyses the synthetic substrate L-prolyl-beta-naphthylamide (Pro-beta NA) was cloned in Escherichia coli. An enzymic plate assay was used to screen for positive clones. The gene, designated pepI, was subcloned into vector pUC18 and sequenced. The nucleotide sequence revealed an 882 bp open reading frame encoding 294 amino acids, coding for an enzyme with a calculated molecular mass of 32883 Da. By cloning under control of the lac promoter the peptidase was highly expressed. Sequence analysis showed that pepI is of a new sequence type, distinct from all peptidases so far sequenced. Amino acid homology to the active site of a Pseudomonas putida esterase and inhibitor studies of the enzyme imply involvement of a serine residue in catalysis.