This article describes a pilot screening program to detect Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) sexually transmitted infections (STIs) in adolescent and adult males newly incarcerated in New York City jails using urine-based nucleic acid amplification technology (NAAT). Between December 8 and 22, 2003, 2,417 males were tested; 162 (6.7%) were found positive for Ct and/or Ng STIs, with 138 (86.8%) exhibiting no STI signs or symptoms and 102 (63%) treated prior to jail release. Younger age, positive urine leukocyte esterase test, and ≥11 recent sex partners were predictors of STI. Urine-based screening and treatment was feasible in this setting and identified STI that would otherwise have been undetected. Jails may thus be important venues for targeted male STI screening.