The gene encoding a 75-kilodalton (kDa) protein of Chlamydia trachomatis was cloned, expressed, and sequenced. Genomic libraries from C. trachomatis serovar D DNA were constructed in vectors pUC18 and lambda gt11 and were screened with a panel of monoclonal antibodies against C. trachomatis antigens. The only recombinants identified were those that reacted with antibody UM-13, which has specificity for a genus-specific epitope on the 75-kDa protein. The gene was localized to a 2.9-kilobase DNA fragment and sequenced. The gene consists of a long open reading frame of 1,956 nucleotides, which translates into 652 amino acids totalling 70,558 daltons in mass. Putative promoter elements and a ribosome binding site were identified within 5'-flanking sequences, and a typical rho-independent terminator was identified within 3'-flanking sequences. Screening of the GenBank nucleic acid sequence data bank revealed extensive similarity between the chlamydial 75-kDa gene and the heat shock protein 70 (hsp70) family or proteins. In particular, 71 and 69% amino acid sequence similarities were identified with hsp70 of Escherichia coli and Bacillus megaterium, respectively. Polyclonal antibodies were produced to the recombinant antigen in rabbits and detected epitopes on elementary bodies in enzyme-linked immunosorbent and indirect microimmunofluorescence assays. Antibodies reacted with an antigen of identical molecular mass in L2 and C serovars in an immunoblot assay and neutralized these serovars in cell culture. The 75-kDa protein appears to be a chlamydial homolog of hsp70, is immunoaccessible on native elementary bodies, and is a target for neutralization.