Staphylococcus lugdunensis is a pathogen of heightened virulence that causes infections resembling those caused by Staphylococcus aureus rather than those caused by its coagulase-negative staphylococcal counterparts. Many types of S. lugdunensis infection, including native valve endocarditis, prosthetic joint infection, and intravascular catheter-related infection, are associated with biofilm etiology. Poly-N-acetylglucosamine (PNAG), a polysaccharide synthesized by products of the icaADBC locus, is a common mechanism of intercellular adhesion in staphylococcal biofilms. Here we report the characterization of ica homologues and the in vitro biofilm formation properties of a collection of S. lugdunensis clinical isolates. Isolates formed biofilms in microtiter wells to various degrees. Biofilm formation by most isolates was enhanced with glucose but diminished by sodium chloride or ethanol. icaADBC homologues were found in all S. lugdunensis isolates tested, although the locus organization differed substantially from that of other staphylococcal ica loci. icaR was not detected in S. lugdunensis, but a novel open reading frame with putative glycosyl hydrolase function is located upstream of the ica locus. icaADBC sequence heterogeneity did not explain the variability in biofilm formation among isolates. PNAG was not detected in S. lugdunensis extracts by immunoblotting with an anti-deacetylated PNAG antibody or wheat germ agglutinin. Confocal microscopy with fluorescently labeled wheat germ agglutinin showed a paucity of PNAG in S. lugdunensis biofilms, but abundant extracellular protein was visualized with SYPRO Ruby staining. Biofilms were resistant to detachment by dispersin B and sodium metaperiodate but were susceptible to detachment by proteases. Despite the genetic presence of icaADBC homologues in S. lugdunensis isolates, PNAG is not a major component of the extracellular matrix of in vitro biofilms formed by this species. Our data suggest that the S. lugdunensis biofilm matrix contains proteinaceous factors.