Many studies indicate that the cell type-specific transcription factor OTF-2 plays a central role in the lymphoid-specific transcription of immunoglobulin genes. In order to better understand the mechanisms regulating transcriptional selectivity, we have initiated a functional dissection of OTF-2. By mutagenesis of an OTF-2 cDNA and by subsequent transfection into non-lymphoid cells we have defined two domains that are required jointly for high levels of transcription from a lymphoid-specific target promoter. One of these domains, located at the extreme carboxy-terminus, is rich in proline. The other domain, which appears to represent a novel type, is located upstream of the POU-specific box and contains clusters of intermingled proline, glutamine and leucine residues. Point mutations that change either groups of glutamine or groups of leucine show that both types of amino acids are required for function. Our data indicate that a high content in a particular amino acid does not necessarily suffice to generate a potent activation domain but that severe structural constraints may also play a major role.