In Acremonium chrysogenum, biosynthesis of cysteine for the formation of cephalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 genomic library in lambdaEMBL3-ble. The cloned DNA fragment encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa that shows great similarity to cystathionine-gamma-lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional because it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-gamma-lyase. The cloned gene did not complement A. nidulans mecA or metG mutants. Enzyme activity assays confirmed that the cloned mecB gene encodes a cystathionine-gamma-lyase activity. The mecB gene is present in a single copy in the wild-type A. chrysogenum (Brotzu's strain) and also in the A. chrysogenum strain C10, a high cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb), as shown by hybridization to A. chrysogenum chromosomes resolved by pulsed-field gel electrophoresis. Transcription of the mecB gene gives rise to a major transcript of 1.5 kb and a minor one of 1.7 kb. The transcript levels were not significantly affected by addition of DL-methionine to the culture, indicating that expression of this gene is not regulated by methionine. The availability of this gene provides a very useful tool for understanding the proposed role of cystathionine-gamma-lyase in splitting cystathionine to supply cysteine for cephalosporin biosynthesis.