Rabbit heart contains two soluble 5'-nucleotidases, termed N-I and N-II, which can be separated using phosphocellulose chromatography. N-I prefers AMP over IMP as substrate, in contrast to N-II which prefers IMP over AMP. Both enzymes require Mg2+, but the optimum Mg2+ concentrations for the two enzymes are different. High concentrations of NaCl inhibit N-I and activate N-II. Purified N-I is activated by ADP but not by ATP. According to Itoh et al. (1986), purified N-II is activated by both ADP and ATP. N-I has been purified approximately 1000-fold to a specific activity of approximately 100 mumol/mg protein/min. The properties of N-I suggest that it is the enzyme responsible for the release of adenosine from AMP under conditions of hypoxia or increased work load.