Many college students are not properly informed about the prevention of sexually transmitted diseases (STDs) and have an inadequate understanding of what constitutes risky sexual behavior. While studies have examined the state of sexual health within young and adult populations, there remains a need for a valid and reliable instrument to measure the extent of STD knowledge in college students. PURPOSE: The purpose of this study was to develop and assess the face validity and internal consistency reliability of an STD knowledge instrument that could serve as a needs assessment and impact evaluation tool at a large university in the south central United States. METHODS: Investigators developed 20 knowledge items per each of the four most common STDs affecting college students: Chlamydia, Gonorrhea, Human Papillomavirus and Genital Herpes. An additional 20 items were developed to test general knowledge regarding STD rates, higher-risk behaviors, and prevention methods. After review of the item pool, investigators agreed upon 12 multiple-choice and/or true/false items per content area, for a total of 60 knowledge questions on the initial instrument. Readability of the instrument was 6.9 on the Flesch-Kincaid Index. The initial instrument was completed by 215 college students, aged 18-25 years, who were recruited from general education courses. RESULTS: Following administration of the instrument, researchers calculated the response frequencies and item total correlations. Items with the lowest total item correlation were discarded until the highest coefficient alpha value was obtained with at least 30 items remaining on the survey. Investigators reviewed the remaining items for face validity. The resulting instrument consisted of 34 knowledge items with acceptable internal consistency reliability (α = 0.78). Individual item-total correlations of the remaining items ranged from 0.11 to 0.45. CONCLUSIONS: Although in its preliminary stages, this STD knowledge instrument has promise as a needs assessment and impact evaluation tool. This instrument should be examined by sexual health experts to assess content validity and administered to a larger sample of college students to enable the researchers to continue exploring the psychometric properties of the instrument.