Insertional mutagenesis with pGhost9::ISS1 resulted in independent insertions in a 350-bp region of the chromosome of Lactococcus lactis subsp. cremoris MG1363 that conferred phage resistance to the integrants. The orientation and location of the insertions suggested that the phage resistance phenotype was caused by a chromosomal gene turned on by a promoter from the inserted construct. Reverse transcription-PCR analysis confirmed that there were higher levels of transcription of a downstream open reading frame (ORF) in the phage-resistant integrants than in the phage-sensitive strain L. lactis MG1363. This gene was also found to confer phage resistance to L. lactis MG1363 when it was cloned into an expression vector. A subsequent frameshift mutation in the ORF completely eliminated the phage resistance phenotype, confirming that the ORF was necessary for phage resistance. This ORF provided resistance against virulent lactococcal phages belonging to the 936 and c2 species with an efficiency of plaquing of 10(-4), but it did not protect against members of the P335 species. A high level of expression of the ORF did not affect the cellular growth rate. Assays for phage adsorption, DNA ejection, restriction/modification activity, plaque size, phage DNA replication, and cell survival showed that the ORF encoded an abortive infection (Abi) mechanism. Sequence analysis revealed a deduced protein consisting of 201 amino acids which, in its native state, probably forms a dimer in the cytosol. Similarity searches revealed no homology to other phage resistance mechanisms, and thus, this novel Abi mechanism was designated AbiV. The mode of action of AbiV is unknown, but the activity of AbiV prevented cleavage of the replicated phage DNA of 936-like phages.