The outer membrane protein OspB of Borrelia burgdorferi, the Lyme borreliosis agent, differs in relative molecular weight (Mr) among strains. To determine whether antigenic variation occurs in B. burgdorferi, a cell population of the human isolate HB19 was cloned first by being diluted in broth and then by being plated on agar medium. Several clones were obtained and characterized by polyacrylamide gel electrophoresis, in situ protease treatment, and Western (immunoblot), Southern, and Northern (RNA) blot analyses. Variants featuring OspB proteins that differed in Mrs and in reactivities with monoclonal antibodies were found. One variant made increased amounts of a 21,000-molecular-weight protein (21K protein) in addition to normal amounts of a 33K OspB protein. Another variant did not produce an OspB protein at all but did express an 18.5K protein. Both the 18.5K and 21K proteins were susceptible in situ to trypsin and were bound by a monoclonal antibody directed against the OspB of strain HB19. There were no differences in the Southern and Northern blot analyses of the different variants. The results led to the following conclusions. (i) Clonal polymorphisms in the surface protein OspB occurred in B. burgdorferi. (ii) Hitherto uncharacterized 18.5K and 21K proteins were protease susceptible, antigenically related to OspB, and apparently produced in greater amounts when an OspB either was not produced or was altered in structure. (iii) The OspB variations, including its absence from cells, were not accounted for by major DNA rearrangements or failure of transcription of the ospB gene.