Aims Identification, cloning, expression and characterization of a novel lipase Lip I.3 from strain Pseudomonas CR-611. Methods and Results The corresponding gene was identified and isolated by PCR-amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0-1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C-terminal type I secretion signal and several putative Ca2+ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca2+ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols. Conclusions A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts. Significance and Impact of the Study The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.