This study sought to determine STI knowledge among high school students and factors associated with such knowledge, and to determine whether poor STI knowledge is associated with chlamydia or gonorrhea infection. Students in an urban United States school district serving a predominantly African American student population participated in a urine-based chlamydia and gonorrhea screening. Participants (N=3563) were surveyed about their knowledge of selected basic facts concerning STIs. Point-scores were assigned to knowledge items. The mean knowledge score was 3.65 (range: 0 to 6; median: 4.00). In a multiple regression analysis, knowledge score was significantly associated with female gender (P<0.001), upper grade level (P<0.001) and a past infection with chlamydia or gonorrhea (P=0.001). In logistic regressions, knowledge score was not significantly associated with current infections with chlamydia (P=0.22) or gonorrhea (P=0.74). There was an insufficient basic knowledge of STIs among students and a lack of association between knowledge and current infections with chlamydia and gonorrhea. Health education curricula taught throughout the high school years should incorporate basic facts concerning STIs.