Adenosine deaminase (ADA) is a well-characterized enzyme involved in the depletion of adenosine levels. A group of proteins with similarity to ADA, the adenosine deaminase-related growth factors (ADGF; known as CECR1 in vertebrates), has been described recently in various organisms. We have determined the phylogenetic relationships of various gene products with significant amino acid similarity to ADA using parsimony and Bayesian methods, and discovered a novel paralogue, termed ADA-like (ADAL). The ADGF proteins share a novel amino acid motif, "MPKG," within which the proline and lysine residues are also conserved in the ADAL and ADA subfamilies. The significance of this new domain is unknown, but it is located just upstream of two ADA catalytic residues, of which all eight are conserved among the ADGF and ADAL proteins. This conservation suggests that ADGF and ADAL may share the same catalytic function as ADA, which has been proven for some ADGF members. These analyses also revealed that some genes previously thought to be classic ADAs are instead ADAL or ADGFs. We here define the ADGF, ADAL, ADA, adenine deaminase (ADE), and AMP deaminase (AMPD) groups as subfamilies of the adenyl-deaminase family. The availability of genomic data for the members of this family allowed us to reconstruct the intron evolution within the phylogeny and strengthen the introns-late hypothesis of the synthetic introns theory. This study shows that ADA activity is clearly more complex than once thought, perhaps involving a delicately balanced pattern of temporal and spatial expression of a number of paralogous proteins.