In Nectria haematococca the MAK1 gene product converts a chick-pea (Cicer arietinum) phytoalexin, maackiain, into a less toxic compound. The presence of MAK1 in this fungal pathogen is also correlated with high virulence on chick-pea. Previous genetic analysis suggested that MAK1 is located on a meiotically unstable, dispensable chromosome. The unstable nature of this chromosome facilitated MAK1 cloning by allowing us to identify a subset of genomic cosmid clones likely to contain MAK1. Truncated forms of the chromosome, generated during meiosis, were isolated from strains either able (Mak+) or unable (Mak-) to metabolize maackiain and used to probe a chromosome-specific cosmid library. Only clones that hybridized exclusively to the chromosome from the Mak+ strain were then screened for their ability to transform a Mak- isolate to the Mak+ phenotype. A 2.7 kb HindIII-PstI fragment was subcloned from a cosmid conferring MAK1 activity, and its nucleotide sequence determined. Because MAK1 transcription is not induced strongly by maackiain, a reverse transcriptase-polymerase chain reaction was required to detect MAK1 transcription in a Mak+ strain, and to isolate MAK1 cDNA fragments. Comparison of the genomic and cDNA sequences of MAK1 revealed the presence of three introns and an open reading frame encoding a protein 460 amino acids in length. Two diagnostic domains in its deduced amino acid sequence suggest MAK1 encodes a flavin-containing mono-oxygenase. MAK1 is the first gene encoding maackiain detoxification to be cloned, and is the second functional gene cloned from this dispensable chromosome. Southern analysis of genomic DNA from ascospore isolates containing MAK2, MAK3, and MAK4 indicated that MAK1 is not homologous to other known maackianin-detoxifying genes.