The cysB gene of A. nidulans was cloned by complementation of a cysB mutation. This is the first cloned eukaryotic genomic sequence coding for cysteine synthase. The gene contains one 71-bp intron and codes for a protein of 370 amino acids. Its N-terminal region has characteristic features of transit peptides, suggesting mitochondrial localisation of the enzyme. The protein shows homology with bacterial and plant cysteine synthases among which it occupies a remote phylogenetic position and apparently represents a distinct subfamily. Transcription of the cysB gene is not appreciably regulated by the concentration of methionine in the growth medium.