The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (alpha and beta) in the metastatic processes, using rat mammary-adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the alpha isoform (nm23-H2 homolog) was more highly expressed than the beta isoform (nm23-H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKalpha or NDPKbeta cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKalpha gene exhibited reduced lung metastasis in a spontaneous metastasis assay.