We have previously identified a cDNA encoding a cellular protein, Tip60 (Tat interactive protein, 60 kDa), that specifically interacts with the Tat (transactivating transcriptional regulator) protein of the human immunodeficiency virus-1 (HIV-1). In this report, we have characterized cellular Tip and find that it is a 60 kDa nuclear protein expressed in a wide variety of differentiated cell lines from insects to man. To identify cellular functions of Tip, we have assayed the effects of Tip on cellular pathways that Tat has been reported to affect. Overexpression of Tip results in an almost complete block in activation of a Gal4-CREB (cAMP response element binding protein) fusion protein by cyclic AMP dependent protein kinase A (PKA). This inhibition appears to be mediated through direct interaction of Tip and CREB, since Tip directly binds to CREB protein in vitro. We show that amino acid substitutions of two conserved amino acids found in the putative acetyl coenzyme A binding motif of Tip completely abolishes the histone acetyltransferase (HAT) activity of recombinant Tip. Inhibition of CREB activation by Tip is not diminished in a HAT negative Tip mutant, indicating that Tip can negatively regulate gene expression independent of HAT activity. Recently, Tip has also been shown to be a transcriptional coactivator of nuclear hormone receptors; therefore, Tip can both activate transcription factors of one signaling pathway (nuclear hormone receptors) and bind to a different transcription factor (CREB) and inhibit activation of another signaling pathway.