Introduction: Gram-negative Aggregatibacter actinomycetemcomitans is recognized as an important periodontal pathogen. A striking property of this bacterium is its ability to form a tenacious biofilm adhering to abiotic surfaces. Both fimbrial and non-fimbrial adhesins are believed to be responsible for this ability. In our study, specific markerless mutants in the biosynthesis genes of cell surface polysaccharides were constructed with the Cre-loxP recombination system to identify non-fimbrial adhesin(s). Methods: Non-fimbriated A. actinomycetemcomitans strain ATCC29523 (serotype a) was used to construct a deletion mutant of serotype-a specific polysaccharide antigen (SPA-a) in lipopolysaccharide (LPS). The LPS was purified through a polymyxin B column following phenol extraction, and verified by silver staining following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblot analysis using rabbit antisera raised against SPA-a. Strains were grown in broth for 2 days and examined for the adherence of bacterial cells on the glass surface. Results: Strain ATCC29523 formed a thin film of bacterial growth on the glass surface. The deletion of SPA-a affected its ability to form this thin film. When this mutant was rescued with the wild-type SPA-a gene cluster, its adherence-positive phenotype was restored. Conclusion: SPA-a in the LPS molecule appears to promote the adherence of A. actinomycetemcomitans cells to abiotic surfaces. © 2008 The Authors.