The outer surface proteins (Osps) of Borrelia burgdorferi, the etiologic agent of Lyme disease, are principle targets of protective immune responses against this organism. Whereas most North American strains of B. burgdorferi in culture express an abundant amount of Osp A, antibodies to this protein are either absent or only weakly detected in the sera of naturally infected patients or experimentally infected mice. In contrast, Osp C, which has variable expression on cultured organisms; elicits an early, strong humoral response. To examine this paradox, we have studied the in vivo adaptation of a cloned population of B. burgdorferi strain N40 during the early course of experimental murine borreliosis. As in human disease, antibodies to Osp A were only weakly present in the early immune repertoire after murine inoculation with low dose (10(3)) spirochetes. In contrast, antibodies to Osp C were prominent, even though on cultured spirochetes Osp C mRNA and protein expression could not be detected by reverse transcription polymerase chain reaction (RT-PCR) or indirect immunofluorescence, respectively. These observations led us to investigate the expression of Osp A and Osp C in vivo. By direct fluorescent staining of uncultured spirochetes ex vivo and by PCR amplification of spirochetal mRNA, we show that Osp C is indeed expressed by some spirochetes after infection in the mouse. Spirochetes expressing Osp A could also be detected within the first 2 wk of infection, but not at 30 d. Osp A mRNA, although present at day 14 of infection, could not be amplified by RT-PCR at day 30, suggesting that the expression of this Osp is transient. This further implies that the late burst in Osp A antibodies in both mice and humans may be anamnestic. These results indicate that either Osp C is upregulated on spirochetes after infection, or Osp C-expressing spirochetes expand preferentially over those expressing Osp A during infection. These results have important implications for vaccine design and offer one explanation for the failure of Osp A antibodies to eradicate spirochetes from the infected host.