Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.