From the lichen Lasallia pustulata a new enzyme, “orsellinate depside hydrolase”, capable of catalyzing the hydrolysis of certain depsides to corresponding resorcylic acid monomers, has been isolated. The enzyme is extremely substrate-specific; only depsides based on orsellinic acid (2,4-di-hydroxy-6-methyl-benzoic acid) alone are acceptable. Phenyl benzoate and m-digallic acid are not hydrolysed. Purification of the enzyme 135-fold has been achieved with a final specific activity of 1300 U/mg of protein. The enzyme has a molecular weight of 42000 as determined by dodecylsulphate-polyacrylamide gel electrophoresis. Only a single sharp peak was obtained by analytical ultracentrifugation. Electrofocussing of the hydrolase in polyacrylamide gel resolved the protein into four distinct enzymatically-active bands. A Km of 56 μM has been determined using the depside lecanoric acid as substrate. The enzyme has been shown to be unusually stable to heat. On treatment of the enzyme for 22 h with 5 mM diisopropylfluorophosphate 75% of the activity was lost, typical of a serine esterase. A spectrophotometric method suitable for the assay of orsellinate depside hydrolase is described.