Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.