Viable but noninfectious (stressed/persistent) chlamydiae are more resistant to azithromycin (AZM) in culture than are organisms in the normal developmental cycle. Chlamydia muridarum-infected mice were exposed to amoxicillin to induce the organisms to enter the persistent/stressed state and subsequently treated with AZM. AZM treatment failure was observed in 22% of persistently infected mice, with an average of 321,667 inclusion-forming units (IFU) shed after AZM treatment. Productively infected mice had a 9% rate of AZM treatment failure and shed an average of 12,083 IFU. These data suggest that stressed chla-mydiae are more resistant to frontline antichlamydial drugs in vivo. C hlamydial species exhibit a unique biphasic developmental cycle, interchanging between the infectious elementary body (EB) and the replicative, noninfectious reticulate body (RB). In-ternalized EBs form vacuoles that fuse to form an inclusion, the membrane-bound structure in which EBs transform to RBs. After multiple rounds of division, RBs condense to form EBs, which are released to infect new host cells. In culture, exposure to environmental insults, including immunological stressors like gamma in-terferon (IFN-) and beta-lactam antibiotics such as amoxicillin (AMX), induces chlamydiae to reversibly detour from this normal developmental cycle (1, 2), entering a noninfectious, viable state alternately termed persistence or the chlamydial stress response. Interestingly, aberrant bodies (ABs) have been observed in vivo in the absence of a known stressor, suggesting that host environmental fluctuations induce at least a small subset of infecting organisms to enter persistence (3). Recently, we demonstrated this state in AMX-stressed Chlamydia muridarum-infected BALB/c mice. In our model, AMX treatment decreased vaginal shedding of infectious chlamydiae by 99% without affecting viability. Shedding of infectious EBs resumed within 1 week after treatment cessation (4). These data demonstrate that AMX can be used to reversibly induce chlamydial persistence in vivo. In culture, persistent chlamydiae are refractory to treatment with first-choice antibiotics. Persistent/stressed C. trachomatis strains within penicillin-exposed Hec1B cells are resistant to treatment with azithromycin (AZM) (5), and IFN-exposure makes C. trachomatis more resistant to doxycycline killing (6). Additionally , doses of AZM or ofloxacin up to 4 the MIC are ineffective against persistent C. pneumoniae infection in culture (7). Thus, many investigators have proposed that persistent chlamydiae may contribute to chronic disease by evading antibiotic treatment. While there is evidence that persistent infection occurs in vivo (8, 9), the absence of an experimentally tractable animal model of viable but noninfectious chlamydial infection has hampered direct testing of this prediction. This study will seek to determine if persistent organisms are resistant to treatment by the first-choice antibiotic, AZM, in our newly characterized mouse model (4). All animal experiments desc...