Transcriptional activation by the herpesvirus protein VP16 (= Vmw65, alpha TIF) is mediated by its C-terminal acidic activation domain. Using GAL4 fusion proteins, we have previously shown that a construct containing two tandem copies of a short eleven amino acid fragment derived from the VP16 domain (DALDDFDLDML, residues 437-447) activates transcription in mammalian cells with an efficiency comparable to a GAL4 fusion with the full VP16 activation domain (residues 413-490). Here we have mutagenized this eleven amino acid core sequence and find that a mutant sequence with little inherent activity can cooperate with a wildtype sequence to yield almost full activity. Moreover, greater activity is observed when the wildtype sequence is positioned at the distal, rather than the proximal, end of the fusion protein, indicating that the distal position facilitates contacts to the transcription apparatus. We have also further reduced the eleven amino acid activating sequence to shorter sequence motifs. Two copies of eight and seven amino acids (DALDDFDL and DDFDLDL, respectively), or four copies of the sequence motif DDFDL are required to reach the activation potential of two eleven amino acid motifs. Four copies of the sequence DDLDL still activate transcription strongly (up to two-thirds of DDFDL), indicating that an aromatic residue is not an essential feature of this type of activation domain. However, repetitions of DDL or DL do not yield activity. Thus the minimal requirement for transcriptional activation is the presence of a sequence of some fifteen to twenty amino acids consisting of a specific array of aspartic acid and leucine residues.(ABSTRACT TRUNCATED AT 250 WORDS)