Babesia microti genomic DNA was purified from parasitized murine erythrocytes, digested with mung bean nuclease and used to construct an expression library in lambda gt11. Polyspecific antisera from mice infected with virulent B. microti organisms (ATCC30221) were used to screen the genomic library for genes encoding major immunogens. High titer antisera selected a recombinant phage, Bm13, containing 3.3 kb of B. microti DNA. Hybridization analysis confirmed the parasite origin of the clone; affinity-purified antibody revealed a native molecular weight of 54,000 for the B. microti protein encoded by the recombinant. Only genomic DNA isolated from the virulent strain of B. microti contained sequences which hybridized to Bm13. Genomic DNA prepared from the Peabody attenuated strain of B. microti or from Babesia bovis DNA did not contain any complementary sequences. These data suggest a possible role for the gene in the virulence of the organism.