The temperate phage P1 encodes two genes whose products antagonize the action of the phage's C1 repressor of lytic functions, namely a distantly linked antirepressor gene, ant, and a closely linked c1 inactivator gene, coi. Starting with an inducible coi-recombinant plasmid, Coi protein was overproduced and purified to near homogeneity. By using a DNA mobility shift assay we demonstrate that Coi protein inhibits the operator binding of the C1 repressors of the closely related P1 and P7 phages. Coi protein (Mr = 7,600) exerts its C1-inactivating function by forming a complex with the C1 repressor (Mr = 32,500) at a molar ratio of about 1:1, as shown by density gradient centrifugation and gel filtration. C1 repressor and Coi protein are recovered in active form from the complex, suggesting that noncovalent interactions are the sole requirements for complex formation. The interplay of repressor and antagonists operating in the life cycle of P1 is discussed.