The TATA box-binding protein-associated factors (TAFs) are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. In hamster ts13 cells, a single amino acid change in TAF(II)250, which disrupts its acetyl-transferase activity at the restrictive temperature, alters the transcription of specific genes involved in cell cycle control. Likewise, disruption of the amino-terminal kinase domain of TAF(II)250 results in transcriptional defects in ts13 cells. However, it was not known whether the acetyl-transferase or kinase domains of TAF(II)250 modulate specific classes of genes and whether these two domains regulate distinct subsets of genes. Here we have used high-density gene-profiling to identify mammalian transcripts that require either the TAF(II)250 acetyl-transferase or protein kinase function for proper expression. We found that transcription of at least 18% of genes are differentially expressed at the restrictive temperature. The promoter region of one of these genes was subsequently characterized, and both upstream elements as well as the core promoter were shown to be TAF(II)250 responsive. We also found that expression of approximately 6% of genes in ts13 cells requires a functional TAF(II)250 amino-terminal kinase domain, but only approximately 1% of these hamster genes also require the TAF(II)250 acetyl-transferase activity. Our results suggest that the two TAF(II)250 enzymatic activities are important for regulating largely nonoverlapping sets of genes involved in a wide range of biological functions in vivo.