Transcriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo.