The immC region of bacteriophage P1 contains the c1 repressor gene and its upstream region with four c1-controlled operators and four open reading frames. A c1 inactivator gene, coi, was defined by mutations in immC that suppress the virulence of the P1virC mutation. The exact location of the coi gene was not known (Scott, J.R. (1980) Curr. Top. Microbiol. Immunol. 90, 49-65). When a variety of P1 immC fragments were inserted into an expression vector, a gene product was inducible for the open reading frame 4 only. We identify this product as the c1 inactivator protein, coi by the following criteria: (a) expression of coi from a recombinant plasmid induces the P1 prophage and inhibits lysogenization of sensitive bacteria by P1; (b) all c1-controlled operator-promoter elements tested in vivo are derepressed by coi; (c) a partially purified coi protein (apparent molecular weight = 4800) interacts with c1 repressor and inhibits its binding to the operator in vitro. Based on these results we refine a model for the regulation of those genes and elements within immC which participate in the decision of P1 to enter the lytic or lysogenic pathway.