Recent experiments in yeast (Saccharomyces cerevisiae) cells have identified a species-specific region of yeast transcription factor IIB (TFIIB) located at residues 144-157. According to the human TFIIB structure, this region is part of a solvent-exposed helix in the first repeat of the carboxyl-terminal core domain. In this report, we systematically analyze four positions in this region (Lys-147, Cys-149, Lys-151, and Glu-152) that together have been shown previously to be important for yeast TFIIB's function in vivo. Our experiments suggest that all of these four positions, and in particular positions 151, 149, and 152, are critical for yeast TFIIB's ability to support cell growth. In addition, we describe an intragenic suppressor screening experiment to identify mutations that reverse, or partially reverse, the temperature-sensitive phenotype of a yeast TFIIB derivative bearing amino acid changes at these four positions to human residues. The suppressor mutations reveal changes at positions 115, 117, and 182 that are located outside the species-specific region of yeast TFIIB, suggesting an extended surface available to interact with other proteins. Finally, we demonstrate that the suppressor mutations restore gene activation in vivo, further supporting the idea that one important function of yeast TFIIB in living cells is to mediate gene activation.