Upton & Buckley (1995) recognized a new family of lipolytic enzymes, which they originally named GDS(L) lipolytic enzymes after the conserved motif containing the catalytic serine. A new name, SGNH hydrolases, was proposed after the four amino acids that are conserved in the active site (Mølgaard et al., 2000). This particular group comprises lipases, proteases, thioesterases, arylesterases, lysophospholipases, carbohydrate esterases and acyltransferases. Some of the SGNH hydrolases display high flexibility in the active site which enables them to accept a wide range of different types of substrates, but it is as yet unclear whether this property of promiscuity is common to all enzymes of this family. We have recently discovered that the extracellular lipase from Streptomyces rimosus (SrLip) exhibits substrate and catalytic promiscuity (Leščić Ašler et al., 2010). In order to gain better insight into the structural and mechanistic basis of enzyme catalysis, we investigated the interactions of this enzyme with several inhibitors. The general mechanism-based serine protease inhibitor 3, 4-dichloroisocoumarin (DCI) was shown to be the most effective and covalently bound to the active-site serine (Zehl et al., 2004). Purified SrLip was incubated with 600-fold molar excess of DCI and crystallisation screens were set. Only one crystal grew from 0.1 M MES pH 6.5, 25% PEG 2000 MME. This crystal was used for data collection on the XRD beamline of the Elettra synchrotron, Trieste, Italy. A 98.4% complete data set was collected to a resolution of 1.7 Å at 100 K. The protein crystallized in the monoclinic space group P21, with unit-cell parameters a = 38.1, b = 78.7, c = 56.6 Å, β= 104.5°. The starting model for the molecular- replacement procedure was built using the Phyre server, based on Phospholipase A1 from Streptomyces albidoflavus NA297. Crystal structure of SrLip will be discussed in detail, with special emphasis on active site shape and topology, considering this enzyme's broad substrate specificity.