This study examined potential facilitators and barriers to pre-exposure prophylaxis (PrEP) use and their association with PrEP acceptability and motivations for adherence among 184 MSM and transgender women living in New York City. Participants were presented with educational information about PrEP and completed a computerized survey. Overall, 55.4% of participants reported willingness to take PrEP. The most highly endorsed barriers to PrEP use were health concerns, including both long-term impacts and short-term side effects, questions about PrEP's impact on future drug resistance, and concerns that PrEP does not provide complete protection against HIV. The most highly endorsed facilitator was free access to PrEP, followed by access to support services such as regular HIV testing, sexual health care/monitoring, and access to one-on-one counseling. Participants of color rated both barriers and facilitators as more important than their White counterparts. In multivariate models, barrier and facilitator scores significantly predicted not only PrEP acceptability, but also motivation for PrEP adherence among those who were likely to use PrEP. PrEP implementation programs should consider addressing these barriers and facilitators in protocol and policy development. Findings underscore the importance of support services, such as sexual health counseling, to the success of PrEP as a prevention strategy.