The AP-1 transcription factor is composed of members of the Fos, Jun and ATF families, and plays a key role in tumor progression. We investigated whether Fos proteins regulate cell motility, and if so, whether this capacity is related to their transactivation potential. Two cell lines with different expression profiles of AP-1 were employed focusing on the Fos-family members c-Fos, Fra-1 and Fra-2. Transactivation motifs are found in c-Fos, but not in Fra-1 or Fra-2. The adenocarcinoma CSML0 cells display a low motility and do not express Fra-1 or Fra-2, and only very little c-Fos. In contrast, the fibroblastoid L929 cells express both Fra-1 and Fra-2, but no c-Fos, and these cells display a high motility. Transfection with Fra-1 or c-Fos, but not with Fra-2, strongly enhanced the motility of CSML0 cells. The effect of Fra-1 required the presence of the N-terminal domain of this protein. Conversely, transfection with a Fos dominant-negative mutant or with anti-sense fra-1 or fra-2, strongly reduced the motility of L929 cells. Changes in cell motility correlated with the morphological appearance and the degree of contact with the substratum. We conclude that Fos proteins have distinct roles in the regulation of cell motility.