TFII-I is a multifunctional phosphoprotein with roles in transcription and signal transduction. Here we report characterization of three additional alternatively spliced isoforms of TFII-I. Employing isoform-specific antibodies, we show that the isoforms form a stable complex in vivo preferentially in the nucleus compared with the cytoplasm. We further show that both homomeric and heteromeric interactions are possible and that the heteromeric interactions between a wild type and a nuclear localization-deficient mutant result in nuclear translocation of the complex, leading us to postulate that complex formation might aid in nuclear translocation. In functional assays all four isoforms individually bind to DNA and transactivate reporter genes to a similar extent. However, although co-expression of different TFII-I isoforms leads to enhanced basal activity, it results in attenuated signal responsive activity. Thus, TFII-I might differentially regulate its target genes via complex or subcomplex formation.