Monoclonal antibodies directed against a 34-kilodalton (kDa) surface immunogen of Treponema pallidum were used to select 12 unique T. pallidum DNA-containing Escherichia coli recombinant clones expressing the recombinant form of the 34-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmids in the host cell. Restriction enzyme analyses and Southern hybridization of plasmid DNA demonstrated that all recombinant clones contained common DNA sequences of T. pallidum origin. Further hybridization analyses revealed that the cloned T. pallidum DNA sequences were an accurate representation of the T. pallidum genomic DNA arrangement. Purified immunoglobulin G (IgG) from pooled immune rabbit serum reacted with the clones, while IgG from pooled normal rabbit serum did not. Results of immunological experiments and Southern hybridization indicated that a similar 34-kDa immunogen was present in T. pallidum subsp. pertenue, but it was absent from four species of nonpathogenic treponemes tested, as well as from homogenates of normal rabbit testicular tissue. Metabolic labeling of the E. coli clones with [35S]methionine followed by radioimmunoprecipitation with monoclonal antibodies revealed that the 35S-labeled recombinant and 125I-labeled native (T. pallidum) forms of the antigen had identical electrophoretic mobilities. The production of a complete antigen by E. coli was independent of the orientation of the foreign gene sequence with respect to vector DNA. T. pallidum also produced an apparently identical immunoprecipitable 34-kDa antigen after metabolic labeling with [35S]methionine in the presence of cycloheximide. The apparent specificity of the 34-kDa immunogen for pathogenic treponemes and its native cell surface association on T. pallidum justifies a more intense study of this antigen and its corresponding gene.