The Tat protein encoded by the human immunodeficiency virus (HIV) is an efficient activator of HIV gene expression. Many eukaryotic transcriptional activators contain a nucleic acid binding domain and a separate activating domain. These activating regions are acidic and often amphipathic. The amino terminus of the HIV-1 Tat protein is acidic with a periodicity of acidic, polar, and hydrophobic residues consistent with that of an amphipathic alpha helix. This region appears to be important for Tat function. We have analyzed the functional significance of acidic residues within the amino-terminal region of Tat by means of site-directed mutagenesis and by testing the capacity of mutant proteins to trans-activate the viral long terminal repeat (LTR) Conservative changes (acidic to acidic) were well tolerated, whereas acidic to neutral and acidic to basic changes markedly reduced Tat activity. The relative importance of each of the three acidic residues correlated with proximity to the amino terminus. Substitution of the entire domain with heterologous sequences that might form an acidic, amphipathic alpha helix partially restored activity when compared with an amino-terminal truncation mutant. In contrast to the observed importance of acidic residues, hydroxylated residues between amino acids 40 and 47 were dispensable for Tat function. These data suggest that the acidity of the amino terminal region is important for Tat function and that Tat-mediated trans-activation may be similar to that of other known activator proteins.