Abstract Functional complementation approach had been used to clone a novel adenosine deaminase gene (ada), a gene that presumably confers Streptomyces antibioticus resistant to 21-deoxycoformycin (21-dCF). Plasmid library containing genomic DNA of S. antibioticus were prepared and transformed into E. coli S๐3834, a strain that had deletion of ada gene and mutation in guaA gene. The transformed colonies were screened for the presence of ada gene by their resistance to ampicillin and their ability to utilize diamino purine (DAP) for growth in minimal medium. Unexpectedly, all of the three positive clones obtained were found to contain the guaA gene than ada gene. In principle, there is equal chance of obtaining either ada or guaA gene with this approach. To test whether functional complementation could be used to clone S. antibioticus ada gene, putative adenosine/AMP deaminase gene from S. coelicolor were amplified by PCR, cloned into pBluescript, and transformed into S๐3834. The transformed cell carrying pBluescript-ADA failed to grow on minimal medium supplemented with DAP. This result suggests that functional complementation may not suitable for cloning of ADA from Streptomyces because substrate specificity of Streptomyces enzyme may be differed significantly from that of E. coli. To over-express and characterize the putative ADA from S. coelicolor, the ada gene was inserted into pET-15b and transformed into BL21(DE3). Protein was purified and characterized. The result indicates the enzyme is a zinc metalloenzyme and could deaminate adenosine and 21-deoxyadenosine but not AMP. Coformycin and 21-dCF whose structures mimic the tetrabedral transition state of the reaction were found to be a potent inhibitor, with K1 of 2.5 x 10-10 and 2.5 x 10-9 M, respectively. Thus, it is proposed that the catalytic mechanism of ADA from S. coelicolor is probably similar to that of the murine enzyme.