An immunological screening strategy was used to select microbial genes expressed only in the host. Differential screening of a Borrelia burgdorferi (the Lyme disease agent) expression library identified a gene (p21) encoding a 20.7-kDa antigen that reacted with antibodies in serum from actively infected mice but not serum from mice immunized with heat-killed B. burgdorferi. Selective expression of p21 in the infected host was confirmed by Northern blot analysis and RNA PCR. Further differential screening of the expression library identified at least five additional B. burgdorferi genes are selectively expressed in vivo. This screening method can be used to identify genes induced in vivo in a wide variety of pathogenic microorganisms for which a gene transfer system is not currently available.