The Wilms' Tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides which differ in the presence or absence of two stretches of amino acids: one of 17 residues (17aa) just N-terminal of the four zinc-fingers and of three residues (K-T-S) between zinc finger 3 and 4. In this study, four human cDNA constructs encoding the Wilms' tumor 1 splice variants were stably transfected into adenovirus-transformed baby rat kidney (Ad-BRK) cells. The in vivo produced WT1 proteins that lacked the KTS residues were found to bind efficiently to both the Egr-1 consensus sequence and the recently described WTE DNA sequence, as determined by electrophoretic mobility shift assays. Our studies show distinct effects of the different WT1 isoforms. Expression of the WT1 (-/+) protein, lacking the 17aa insert, strongly suppressed the tumorigenic phenotype of the Ad-BRK cells. Intriguingly, expression of the WT1 (-/-) protein, lacking both inserts, increased the tumor growth rate. In contrast to the growth in vivo, the growth rate of the transfectants in tissue culture is not influenced by any of the WT1 isoforms. However, the suppression of tumorigenicity appears to be correlated with a reduced ability of the cells to grow in serum-free medium.