The FDA approval of emtricitabine/tenofovir disoproxil fumarate for pre-exposure prophylaxis (PrEP) in 2012 has raised questions about the delivery of PrEP in a real-world setting. iPad-based questionnaires were given to providers at conferences in California and New York to assess knowledge, experience and attitudes regarding PrEP in HIV and non-HIV providers. HIV provider status was defined either by self-identification or by having greater than 5 years of HIV care experience. Knowledge scores were the sum of correct answers from five PrEP knowledge questions. Univariate analyses used t-test to compare knowledge scores and Fisher's exact test for past or future PrEP prescription between HIV and non-HIV providers. Multivariable linear or logistic regression models were used to assess factors associated with the outcomes. Of 233 respondents, the mean age was 40 years, 59 % were White, 59 % were physicians and 52 % were HIV providers. In univariate analysis, mean PrEP knowledge scores (max 5) were significantly higher for HIV providers (2.8 versus 2.2; p < 0.001), age > 41 (mean 2.8 versus 2.3; p = 0.004), White race (2.7 versus 2.2; p = 0.026) and participants in the New York region (3.0 versus 2.3; p < 0.001). In a multivariable model of knowledge scores, all but age remained significant. Among 201 potential prescribers, the rate of prior PrEP prescription was higher among HIV providers than non-HIV providers (34 versus 9 %; p < 0.001) and by knowledge score, but the association with provider status was no longer significant in multivariable analysis that controlled for knowledge. Intent to prescribe PrEP in the future was high for all provider types (64 %) and was associated with knowledge scores in multivariable analysis. The most common concerns about PrEP (>40 % of providers) were drug toxicities, development of resistance and patient adherence to follow-up; 32 % identified risk compensation as a concern. HIV providers had significantly greater PrEP knowledge than non-HIV providers, but differences by provider type in past PrEP prescription were largely dependent on knowledge. Future PrEP prescription was also associated with knowledge, though all providers expressed greater future use. Education of potential PrEP providers will be a key component of successful PrEP implementation.