Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in-frame start codon. Another open reading frame, icd, 5' upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene-carrying plasmids we have overproduced and purified Ant1/2 in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N-terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N-terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co-precipitate with the Ant1/2 complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1-inactivating function by a direct protein-protein interaction.