Adenosine production catalysed by cytosolic 5'-nucleotidase (cN-I) regulates diverse physiological processes. We report here a mouse cN-I (mcN-I) cloned from heart and testis. The open reading frame contains several potential translation initiation sites, which yield similarly active 5'-nucleotidases. Using overexpression in COS-7 cells we showed that mcN-I, like the previously cloned pigeon cN-I, is activated by ADP and catalyses adenosine formation during ATP breakdown. The N- and C-termini of mcN-I and pcN-I are divergent. Deletion of the 12 C-terminal amino acids or the first 19 N-terminal amino acids of pcN-I does not diminish activity, although deletion of the first 31 N-terminal amino acids reduces activity by 70%. Overall mcN-I is only 66% identical to pcN-I or the recently cloned human cN-I (hcN-I), while hcN-I and pcN-I are 85% identical. We report here a partial hcN-I sequence that is only 70% identical with the published hcN-I amino acid sequence but is 87% identical with mcN-I. Both hcN-I sequences have perfect matches to distinct human genome sequences. Our data imply the existence of at least two genes for cN-I, cN-I(A), previously cloned from pigeon and human, and cN-I(B) that we report here from mouse and partially from human.