Fungi secrete many different enzymes to deconstruct lignocellulosic biomass, including several families of hydrolases, oxidative enzymes, and many uncharacterized proteins. Here we describe the isolation, characterization, and primary sequence analysis of an extracellular aldonolactonase from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile). The lactonase is a 48-kDa glycoprotein with a broad pH optimum. The enzyme catalyzes the hydrolysis of glucono-δ-lactone and cellobiono-δ-lactone with an apparent second-order rate constant, k(cat)/K(m), of ~1 × 10(6) M(-1) s(-1) at pH 5.0 and 25°C but is unable to hydrolyze xylono-γ-lactone or arabino-γ-lactone. Sequence analyses of the lactonase show that it has distant homology to cis-carboxy-muconate lactonizing enzymes (CMLE) as well as 6-phosphogluconolactonases present in some bacteria. The M. thermophila genome contains two predicted extracellular lactonase genes, and expression of both genes is induced by the presence of pure cellulose. Homologues of the M. thermophila lactonase, which are also predicted to be extracellular, are present in nearly all known cellulolytic ascomycetes.