Previous work has shown that the sieA gene of Salmonella bacteriophage P22 is located between the genes mnt and 16. We cloned DNA fragments of the region into multicopy vectors and tested the transformants for mediating superinfection exclusion. Subcloning, phenotypical tests, and DNA sequencing resulted in the identification of the sieA gene. There are two possible initiation codons within one open reading frame of 492 or 480 bp. The deduced amino acid sequence leads to a hypothetical polypeptide with a calculated molecular mass of 18.8 or 18.3 kDa, respectively. According to three hydrophobic regions, all of which are long enough to span the membrane, the product of sieA should be a protein of the inner membrane of a P22-lysogenic cell of Salmonella typhimurium. The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction.