An HIV transmission prevention program incorporating universal voluntary counseling and testing (UVCT) was conducted in a general practice (GP) clinic of a Thai hospital. Of the 494 participating patients, 356 (72%) accepted HIV-UVCT. Independent factors associated with HIV-UVCT acceptance included participating in the program after office hours (4-8 pm; P < .001), living with domestic partner with no marriage (P = .01), and having primary school education or less (P = .02). The main reasons for declining HIV-UVCT were time constraint (38%) and perceiving self as no risk (35%). Among the 356 patients undergoing HIV-UVCT, having moderate to high HIV risk (P < .001) and male sex (P = .01) were independently associated with low HIV risk perception. By HIV-UVCT, the rate of new HIV infection was 4 (1.1%) of 356 patients. Of these 4 newly diagnosed HIV-infected patients, 3 (75%) were homosexual men. The findings suggest feasibility of HIV-UVCT in our GP clinic and factors to be considered for improving the program.