Preliminary experimentation has suggested that a research-use-only real-time transcription-mediated amplification assay for Treponema pallidum (RUO T. pallidum TMA) yields instances of T. pallidum nucleic acid detection that coincide with non-treponemal serology in men who have sex with men (MSM) at increased risk for sexually transmitted infection. To further characterize the specificity of RUO T. pallidum TMA testing, 3,586 rectal swab specimens reported as "not detected" by the assay generated a mean endpoint FAM fluorescence of 637.5 units. Introduction of Treponema denticola, Treponema phagedenis, and Treponema refringens nucleic acid into matrices generated mean endpoint FAM fluorescence ranging from 620.7 to 633.5 units (P ≥ 0.15 versus control). Introduction of lubricant to a pooled rectal swab matrix did not result in elevated FAM fluorescence (mean endpoint value 628.3 units [95% CI 612.0, 644.6]; P = 0.67). Introduction of talcum powder, urine, seminal fluid, and blood also failed to generate increased FAM fluorescence (P ≥ 0.20). Analytic sensitivity assessment was measured by serial 10-fold dilution of T. pallidum whole organism or in vitro 23S rRNA transcript in specimen transport medium or pooled rectal swab matrix and interrogation by the assay. Probit analysis estimated sensitivity (95% detection) of RUO T. pallidum TMA at 421-5,707 in vitro transcript copies/mL and 9-48 T. pallidum cells/mL, depending on dilution matrix. These results support RUO T. pallidum TMA as a highly sensitive method for T. pallidum detection that is not impacted by potentially cross-reactive organisms or interfering substances and may have adjunctive diagnosis capability for syphilis in MSM. IMPORTANCE: Research-use-only Treponema pallidum transcription-mediated amplification (RUO T. pallidum TMA) has the potential to improve laboratory diagnosis of syphilis, particularly in patients at increased risk for sexually transmitted infection. The high analytic sensitivity and lack of cross-reactivity of the assay can facilitate other laboratories exploring the use of the test in a research setting.