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T here is now substantial evidence that Borrelia burgdorferi, the Lyme disease spirochete, undergoes major alterations in antigenic composition as it cycles between its arthropod and mammalian hosts. In this report, we cultivated B. burgdorferi 297 within dialysis membrane chambers implanted into the peritoneal cavities of rats to induce antigenic changes similar to those which occur during mammalian infection. Chamber-grown spirochetes, which remained fully virulent, did not express either outer surface protein A or Lp6.6, lipoproteins known to be downregulated after mammalian infection. However, they did, express p21, a well characterized outer surface protein E homologue, which is selectively expressed during infection. SDS-PAGE, two-dimensional gel electrophoresis, and immunoblot analysis revealed that chamber-grown borreliae also expressed uncharacterized proteins not expressed by in vitro-cultivated spirochetes; reactivity with sera from mice chronically infected with B. burgdorferi 297 confirmed that many of these novel proteins are selectively expressed during experimental murine infection. Finally, we used differential display RT-PCR to identify transcripts of other differentially expressed B. burgdorferi genes. One gene (2.9-7lpB) identified with this technique belongs to a family of genes located on homologous 32- and 18-kb circular plasmids. The lipoprotein encoded by 2.9-7lpB was shown to be selectively expressed by chamber-grown spirochetes and by spirochetes during experimental infection. Cultivation of B. burgdorferi in rat peritoneal implants represents a novel system for studying Lyme disease spirochetes in a mammalian host-adapted state.