<p>HIV prevention efforts have traditionally focused on urban areas, yet about one-fourth of new HIV diagnoses in the U.S. are in non-urban areas. This study explored rural and urban differences in perceived HIV risk; perceived HIV stigma; and pre-exposure prophylaxis (PrEP) awareness, attitudes, beliefs, communication behaviors, and use to inform the development of communication messages to promote informed decision-making among available HIV prevention options, including PrEP. We conducted interviews, preceded by a brief survey, with 255 adults in 5 rural and 6 urban locations throughout the U.S. with high HIV burden. Participants from rural areas more frequently described their risk of getting HIV as low compared with those from urban areas, although partly due to differences in gender/sexual identity and sexual risk. Participants from rural areas more frequently reported perceived stigma around getting tested for HIV, taking PrEP to prevent HIV, or having HIV and less frequently reported having heard of PrEP and having a healthcare provider talk with them about PrEP compared with those from urban areas. No participants from rural areas reported using PrEP, although 48% of those with HIV-negative or unknown status were at substantial risk based on reported risk factors. Our findings highlight notable differences in perceived HIV risk; perceived HIV stigma; and PrEP awareness, attitudes, beliefs, communication behaviors, and use between individuals residing in rural and urban areas, suggesting that HIV prevention messaging needs to be tailored for rural audiences to support receptivity.</p>