The metastasis suppressor NM23-H1 possesses 3 enzymatic activities in vitro, a nucleoside diphosphate kinase (NDPK), a protein histidine kinase and a more recently characterized 3′-5′ exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3′-5′ exonuclease to metastasis suppressor function has not been addressed in detail. To this end, site-directed mutagenesis and biochemical analyses of bacterially expressed mutant NM23-H1 proteins have identified mutations that disrupt the 3′-5′ exonuclease alone (Glu5 to Ala, or E5A), the NDPK and histidine kinase activities tandemly (Y52A, H118F) or all 3 activities simultaneously (K12Q). Although forced expression of NM23-H1 potently suppressed spontaneous lung metastasis of subcutaneous tumor explants derived from the human melanoma cell line 1205LU, no significant metastasis suppressor activity was obtained with the exonuclease-deficient variants E5A and K12Q. The H118F mutant, which lacked both the NDPK and histidine kinase while retaining the 3′-5′ exonuclease, also exhibited compromised suppressor activity. In contrast, each mutant retained the ability to suppress motility and invasive characteristics of 1205LU cells in culture, indicating that the NM23-H1 molecule possesses an additional activity(s) mediating these suppressor functions. These studies provide the first demonstration that the 3′-5′ exonuclease activity of NM23-H1 is necessary for metastasis suppressor function and further indicate cooperativity of the 3 enzymatic activities of the molecule on suppression of the metastatic process.