We found that Acinetobacter baumannii contains a pgaABCD locus that encodes proteins that synthesize cell-associated poly-beta-(1-6)-N-acetylglucosamine (PNAG). Both a mutant with an in-frame deletion of the pga locus (S1Deltapga) and a transcomplemented strain (S1Deltapga-c) of A. baumannii were constructed, and the PNAG production by these strains was compared using an immunoblot assay. Deleting the pga locus resulted in an A. baumannii strain without PNAG, and transcomplementation of the S1Deltapga strain with the pgaABCD genes fully restored the wild-type PNAG phenotype. Heterologous expression of the A. baumannii pga locus in Escherichia coli led to synthesis of significant amounts of PNAG, while no polysaccharide was detected in E. coli cells harboring an empty vector. Nuclear magnetic resonance analysis of the extracellular polysaccharide material isolated from A. baumannii confirmed that it was PNAG, but notably only 60% of the glucosamine amino groups were acetylated. PCR analysis indicated that all 30 clinical A. baumannii isolates examined had the pga genes, and immunoblot assays indicated that 14 of the 30 strains strongly produced PNAG, 14 of the strains moderately to weakly produced PNAG, and 2 strains appeared to not produce PNAG. Deletion of the pga locus led to loss of the strong biofilm phenotype, which was restored by complementation. Confocal laser scanning microscopy studies combined with COMSTAT analysis demonstrated that the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by wild-type and pga-complemented A. baumannii strains were significantly greater than the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by the S1Deltapga mutant strain. Biofilm-dependent production of PNAG could be an important virulence factor for this emerging pathogen that has few known virulence factors.