Most organisms appear to have a molybdenum cofactor consisting of a complex of molybdenum and a pterin derivative. Very little is known about molybdenum cofactor biosynthesis in plants or other eukaryotes, because the instability of the cofactor and its precursors makes it difficult to analyze this pathway. We have isolated two cDNA clones from the higher plant Arabidopsis thaliana encoding genes involved in early steps of molybdenum cofactor biosynthesis. The cDNAs were obtained by functional complementation of two Escherichia coli mutants deficient in single steps of molybdenum cofactor biosynthesis. The two cDNAs, designated Cnx2 and Cnx3, encode proteins of 43 and 30 kDa, respectively. They have significant identity to the E. coli genes, moaA and moaC, involved in molybdenum cofactor biosynthesis. Both genes have N-terminal extensions that resemble targeting signals for the chloroplasts or the mitochondria. Import studies with the translated proteins and purified mitochondria and chloroplasts did not show import of these proteins to either of these organelles. Northern analysis show that Cnx2 is expressed in all organs and strongest in roots. Cnx3 is not expressed in abundant levels in any tissue but roots. For both genes there is no detectable difference in the expression level from plants grown with nitrate or with ammonium. The Cnx2 gene has been mapped to chromosome II. Southern analysis suggests that both genes exist as single copies in the genome.