Deregulated expression of genes is found in cancer cells, which may affect malignant properties, but it is unclear whether such modulation occurs allele-specifically. This study shows that the gene coding alpha4 integrin, a cell adhesion molecule, underwent allelic inactivation in a series of heterozygous murine fibrosarcoma cell lines (MST lines) with different metastatic potentials. P4 cells expressed the alpha4 integrin gene from one allele at a level comparable to that of the primary MST1 tumor, whereas the descendent lines of P4 exhibited decreased expression of both alleles. No allelic loss of DNA was observed in these cells. Other four clones derived from P4 and five clones from a different tumor also showed such two-step inactivation. Intriguingly, the loss of expression was correlated with the acquisition of spontaneous, but not artificial, metastatic ability. This is consistent with the previous result of inverse relation between the expression of alpha4/beta1 integrin and the invasive potential of B16 melanoma cells. Analysis of DNA methylation and chromatin state of the alpha4 integrin gene failed to provide a clue to difference between the two alleles in the cell lines. These results suggest that the allelic inactivation is a process giving loss of function to one allele, although the mechanism is unclear.