The success of the proposed HIV testing and counselling (HTC) initiative among learners at high school in South Africa depends on their acceptability of the service, which may be influenced by their HIV/AIDSknowledge, perceptions, and attitudes. The study assessed high school learners’ HIV knowledge and attitudes towards learners infected with HIV. The survey was part of formative evaluation with grade 10-12 learners in order to assess the acceptability of HTC at schools. The study consisted of 17 high schools in two provinces in South Africa. T- tests were used to compare the differences in the mean HIV knowledge scores. Logistic regressions were performed to establish relationships between demographic variables, attitudes, and HIV knowledge scores. The sample consisted of 2970 learners aged 14-27years, 1810 (61%) had had sex, and 1494 (50.3%) had tested for HIV in the past year. The majority had high knowledge scores: 87% on HIV-related knowledge, 98.6% on the modes of transmission, and 73% on prevention. Overall, HIV knowledge was high, with a total mean score of 20.5 out of 26 points. There were significant differences in the HIV knowledge total mean scores; females had higher scores (M= 20.6, SD=2.3) than males (M=20.3, SD=2.4), and the younger age group (M= 20.5, SD=2.4) had higher scores than the older age group (M=20.1, SD=2.1). A quarter (25.4%) exhibited negative attitudes, 13.9% felt that HIV-positive learners should be isolated, and 75% would not date HIV-positive learners. Attitudes were associated with gender (OR=0.48, CI: 0.41-0.57), grades (OR=3.24, CI: 2.36-4.44), and total HIV knowledge scores (OR=2.21, CI: 1.43-3.43). Despite substantial HIV knowledge, there were knowledge gaps and misconceptions, which resulted in negative attitudes towards the disease. Appropriate sexual health education and HIV prevention interventions in school are crucial in correcting the knowledge gaps as well as reducing stigma and discrimination to assist learners to be accepting of HIV-positive persons.