Hybrid pBR322 plasmid clone banks comprised of more than 125,000 recombinant DNA clones and representing the entire Treponema pallidum Nichols genome were constructed in Escherichia coli K-12 RR1. The two clone banks individually contain over 53,000 and 72,000 recombinant clones. The number average and mass average sizes of the cloned DNA inserts were found to be approximately 12 and 13 kilobase pairs, respectively, indicating the presence of large treponemal DNA inserts in a majority of recombinant clones. To detect E. coli clones synthesizing T. pallidum antigens as hybrid plasmid gene translation products in the clone bank, a simplified, direct, solid-phase radioimmuno-colony blot (RICB) assay was developed employing immunoglobulin G antibody isolated from anti-T. pallidum immune rabbit serum. Clones with positive reactivities in the RICB assay were isolated at frequencies of 0.1 to 0.2%. One isolated RICB-positive clone, designated RICB2-1, produced a very strong signal in the RICB assay and was subsequently found through E. coli cell-free in vitro transcription/translation analysis to encode the synthesis of two gene translation products with apparent molecular weights of 77,000 and 44,000. The 44,000-dalton protein was effectively immunoprecipitated from [35S]methionine-labeled E. coli clone cells by using either immune rabbit serum (preabsorbed with Treponema phagedenis biotype Reiter antigens) or selected human syphilitic serum, whereas the 77,000-dalton protein was never immunoprecipitable by similar methods. Purified plasmid DNA from clone RICB2-1 contained a treponemal DNA insert of 3.70 kilobase pairs, which was of suitable size to code for the 121-dalton (44 + 77) protein. The insert was also flanked on each end by PstI sites and possessed three internal PstI sites with fragment sizes of 2.15, 1.18, 0.20, and 0.17 kilobase pairs. Purified clone RICB2-1 plasmid DNA was capable of transforming recipient E. coli cells to virtually 100% RICB reactivity, thus substantiating the plasmid-encoded characteristic. Further experiments employing various antisera in radioimmunoprecipitation systems utilizing cell-free in vitro synthesized gene translation products from clone RICB2-1 also provided the first evidence that E. coli may be capable of using endogenous T. pallidum DNA promotors for genetic expression. These studies, amplified by the isolation of a potentially significant immunoprecipitable 44,000-dalton recombinant protein antigen, point to the importance of the "cloned antigen gene" approach for the eventual eludication of specific antigens or immunogens operative in the pathogenesis, immunology, and serodiagnosis of T. pallidum infection.