An activator protein, Alt, synthesized during the early state of lytic infection is required for transcription of the late operon in the lactococcal phage TP901-1. In order to identify amino acid residues in the Alt protein required for activation of the TP901-1 late promoter, Plate, hydroxylamine mutagenesis was performed, resulting in almost saturating mutagenesis of alt. Twenty-three different non-functional alt alleles containing one, and in one case two amino acid exchanges were isolated and analyzed. Eight of the twenty-three mutant proteins were still able to activate the Plate promoter to some extent. Our results show that alt encodes a protein of 16.7 kDa and that the last fourteen amino acids in the C-terminal part of the protein are required for activation of the Plate promoter. By combining sequence analysis with experimental data we suggest that the C-terminal half of the Alt protein contains a helix-turn-helix-like motif involved in DNA binding. We also propose that the C-terminal half of the Alt protein may be involved in interactions with the bacterial RNA polymerase, whereas the N-terminal half of the protein is proposed to be important for the overall protein structure.