Early studies of eye pigmentation in Drosophila melanogaster provided compelling evidence that the cinnabar (cn) gene encodes the enzyme kynurenine 3-monooxygenase (EC 220.127.116.11). Here we report the cloning of approximately 60 kb of DNA encompassing the cn gene by chromosome walking in the 43E6-F1 region of chromosome 2. An indication of the position of cn within the cloned region was obtained by molecular analysis of mutants: 9 spontaneous cn mutants were found to have either DNA insertions or deletions within a 5 kb region. In addition, a 7.8 kb restriction fragment encompassing the region altered in the mutants was observed to induce transient cn function when microinjected into cn- embryos. The cn transcription unit was identified by Northern blotting and sequence analysis of cDNA and genomic clones from this region. The predicted cn protein contains several sequence motifs common to aromatic monooxygenases and is consistent with the assignment of cn as encoding the structural gene for kynurenine 3-monooxygenase.