Abstract Objectives: In 2009, the American College of Obstetricians and Gynecologists (ACOG) issued new guidelines for cervical cancer screening to begin at age 21 based on the low incidence of cancer and the adverse effects associated with follow-up of abnormal screening results in young women. It is unclear whether low-income, minority adolescent girls, often presenting with higher risk for human papillomavirus (HPV) infection and cervical cancer, are screened according to current guidelines. In this study, we examine rates of first lifetime Papanicolaou (Pap) test and predictors of first screening at <21 years of age among adolescents within a large ambulatory care network serving an urban, low-income, minority population in New York City. Methods: We compared rates of first lifetime Pap test between June 2007-November 2009 (pre-guideline) and December 2009-June 2012 (post-guideline). Data were obtained from electronic health records of adolescent females (ages 13-20) who had at least one clinic visit within the ambulatory care network during each study period and no Pap tests before each study period (pre-guideline: n=7,700; post-guideline: n=9,637). Clinic sites included pediatric, family medicine, family planning, obstetrics, gynecology, or school-based health facilities. We examined screening rates in each study period by demographic characteristics, health care factors, sexually transmitted infection (STI) testing history, and HPV vaccination status. We employed multivariable regression models to identify demographic and health care factors associated with receiving a first lifetime Pap test at <21 years of age (over-screening) in the post-guideline period. Results: Rates of first lifetime Pap test declined from 19.3% to 5.7% (p <0.001) between the two study periods. Demographic, health care, and STI testing characteristics of adolescent girls were similar for the two study periods. The average age for each study period was 15 years. The majority of girls were Latina (pre-guideline: 75.8%, post-guideline: 76.8%) and most were covered by public insurance (pre-guideline: 69.1%, post-guideline: 72.7%). Only HPV vaccination history differed between the two periods, with higher initiation and completion rates among girls in the post-guideline period. Over-screening remained high in the post-guideline period for adolescent girls without insurance (9.9%), those who visited gynecologic, obstetrics, or family planning clinics (9.6%), had five or more clinic visits during the study period (11.3%) or were recently tested for a sexually transmitted infection (7.3%). Adjusted multivariable logistic regression results showed receiving care from gynecology, obstetrics, or family planning clinics (OR 3.87, 95% CI: 2.65-5.65) compared to pediatric clinics, having more clinic encounters (OR 6.72, 95% CI: 4.77, 9.45), and older age (OR 4.72, 95% CI: 2.97, 7.49) were associated with over-screening in the post-guideline period. HPV vaccination status, language spoken, and race/ethnicity were not associated with over-screening. Conclusions: While rates of first lifetime Pap tests declined substantially following the 2009 guidelines, we found that adherence to new recommendations differed by health care system factors, including site of care, insurance status, and number of clinic encounters. It is important to eliminate unnecessary medical tests, especially if disclosure and follow-up of abnormal screening results can lead to other adverse effects. In the quickly evolving field of cervical cancer control, it will be important to monitor practice trends as they relate to shifts in population-based guidelines, especially in relation to high-risk populations. Citation Format: Jennifer Tsui, Annika Hofstetter, Karen Soren. Cervical cytology screening patterns among low-income, minority adolescents in New York City following the 2009 ACOG guidelines. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C27.