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Introduction Experimentally induced cervical chlamydial infection in the macaque may naturally cross-infect rectal epithelial cells which are also prone to Chlamydia trachomatis (CT) infection. This would be a significant finding in the field of STI preventive strategies, particularly when products intended for vaginal use are assessed for efficacy. If cross-infection does occur, it will be important to specifically assess rectal secretions for evidence of infection in vaginal product efficacy studies. Methods Twelve pigtailed macaques underwent direct cervical inoculation (1 mL C. trachomatis serovar E; 5E6IFU), followed by five weekly exams to detect infection in cervical and rectal secretions. Inoculant was delivered to the face of the cervix/vaginal fornix via 1 mL tuberculin syringe. Secretions were collected on dacron swabs. Chlamydial infection was detected at each site by culture and by nucleic acid amplification (NAAT: Aptima2) assays. Results Ten of twelve macaques tested positive for cervical chlamydial infection by culture and NAAT assays. The other two were transiently positive (2 weeks, 1 week) by NAAT only. All but three animals had chlamydial rRNA amplified from rectal swabs on at least one occasion. Five animals remained NAAT positive in rectal secretions for three weeks or more. One of these macaques had replicating chlamydia cultured from a rectal swab (week 3 only), followed by a spike in culture positivity from her next cervical sample (week4). Conclusion Experimental CT infection of the cervix indeed gave rise to chlamydia detection in rectal secretions in the majority of animals in this study. Culture positivity in cervical samples did not predict chlamydia detection in rectal samples. The paucity of culture positive results from rectal samples may be related to faecal contamination. Clearly it is advisable that rectal secretions be assessed for evidence of chlamydia in studies designed to assess prevention/treatment of cervical CT infections. Disclosure of interest statement This research was supported by NIH Contract Number HHSN272201000006I, Task Number HHSN27200008 and by the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through Grant Number P51 OD010425 Washington National Primate Research Centre. No pharmaceutical grants were received in the development of this study.