Adenosine increases blood flow and decreases excitatory nerve firing. In the heart, it reduces rate and force of contraction and preconditions the heart against injury by prolonged ischemia. Based on indirect kinetic arguments, an AMP-selective cytosolic 5'-nucleotidase designated cN-I has been implicated in adenosine formation during ATP breakdown. The molecular identity of cN-I is unknown, although an IMP/GMP-selective cytosolic 5'-nucleotidase (cN-II) and an ecto-5'-nucleotidase (e-N) have been cloned. We utilized the high abundance of cN-I in pigeon heart to purify a 40-kDa subunit for partial protein sequencing and subsequent cDNA cloning. We obtained a full-length clone encoding a novel 40-kDa peptide, unrelated to cN-II or e-N, that was most abundant in heart, brain, and breast muscle. Immunolocalization in heart showed a striated cytoplasmic location, suggesting association with contractile elements. Transient expression in COS-7 cells, generated a 5'-nucleotidase that catalyzed adenosine formation from AMP, which was increased during ATP catabolism. In conclusion, the cloning and expression of cN-I provides definitive evidence of its ability to produce adenosine during ATP breakdown.