A component of the cell walls of certain enteric bacteria has been identified that cross-reacts with an HLA-B27-associated cell-surface structure on lymphocytes and other cell types from patients with ankylosing spondylitis. This component, or "modifying factor," from one particular organism, Klebsiella K43-BTS1, has been studied in detail. A purification scheme has been developed using preparative electrofocusing and gel-permeation high performance liquid chromatography techniques and the purified material used in various characterization studies. A previous study demonstrated that the modifying factor has an approximate molecular weight of 30,000 and an isoelectric point of 5.4-5.5. In this study two-dimensional gel electrophoresis experiments demonstrated that the modifying factor is associated with a single protein component of the cell wall of this organism. Pronase and papain destroyed the modifying factor activity whereas trypsin and alpha-chymotrypsin degraded the factor into smaller fragments without destroying its ability to modify B27+ AS- lymphocytes. Neuraminidase did not affect the modifying factor itself but did affect B27+ AS- lymphocytes such that they became unresponsive to modification. Sugar inhibition studies suggested that sugar groups are probably not involved in the function of the modifying factor. The availability of purified modifying factor should permit more detailed chemical analyses as well as functional studies to determine the significance of this molecule to the pathogenesis of ankylosing spondylitis.