Non-invasive, non-metastatic mouse BW5147 T-lymphoma cells were treated with non-mutagenic concentrations of the hypomethylating agent 5-azacytidine (5-aza-C). Subsequently, invasive variants were selected on monolayers of rat embryo fibroblasts. The estimated frequency of induction of invasive variants was smaller than 1 in 10(6) cells. We obtained several independent clones that were stable in the expression of the invasive phenotype. In contrast to the parental cell line, the highly invasive clones produced widespread metastases upon tail vein injection in all the syngeneic AKR mice tested, whereas clones with an intermediate level of invasiveness formed metastases only in part of the mice tested. DNA analysis using the methylation-sensitive and insensitive restriction enzymes, Hpa-II and Msp-I, respectively, showed that the DNA of the invasive variants remained hypomethylated, up to 6 months after 5-aza-C treatment. 5-aza-C is thus able to induce invasive and metastatic potential in the BW5147 T-lymphoma cells, similar to the activated human c-Ha-ras oncogene or human chromosome 7, as studied previously. The acquisition of invasive and metastatic potential is presumably caused by DNA hypomethylation and thus activation of one or more silent invasion controlling genes.