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Substance abuse treatment centers provide an opportunity to offer sexually transmitted infection (STI) screening to a high-risk and hard-to-reach population.The goal was to assess STI prevalence, risk factors, and acceptability of STI screening among females at substance abuse treatment centers with use of urine testing by ligase chain reaction and self-collected swab specimens.Adult, female inpatients were offered free testing and treatment for chlamydia infection, gonorrhea, and trichomonas infection. Interviews were conducted to collect risk behavior data.Eighty-six percent of inpatients (180/209) accepted testing. Twenty-three percent (41/177) had an STI. Of those with an STI, 90% (37/41) had trichomonas infection. All 41 infected patients received treatment. Drug use before sex, exchange of sex for money/drugs, and any gynecological complaint were significantly associated with infection. Most women were uninsured (76%). Only 45% had undergone a medical examination in the past year.STI screening is highly acceptable among women in substance abuse treatment centers. Substance users are at high risk for STIs and may not otherwise receive medical care.