This study sought to establish whether there were measurable differences in the level of knowledge about HIV/AIDS between hearing individuals and individuals who identified themselves as deaf sign language users in Swaziland. A cross-sectional survey of 191 rural and urban hearing and deaf adults was undertaken in Swaziland in December 2003. A structured questionnaire was administered, seeking to establish whether there were statistically significant differences between hearing and deaf populations in their level of knowledge about HIV/AIDS symptoms, transmission and prevention, as well as differences in sources of information about HIV/AIDS. Additional questions were asked regarding whether there were differences in accessibility of HIV testing services and HIV/AIDS-related healthcare for the two groups. Significant differences in levels of knowledge about HIV/AIDS were identified between the hearing and deaf respondents. The deaf population was significantly more likely (P<0.05) to believe in incorrect modes of HIV transmission (e.g. hugging and airborne transmission) and HIV prevention (e.g. avoiding sharing utensils and eating healthy foods). Almost all of the deaf respondents (99%) reported difficulties in communicating with healthcare facility staff, which may result in less use of HIV voluntary counseling and testing services. This paper reports the results of this study, and discusses the need for targeted HIV/AIDS education campaigns and improved accessibility in healthcare facilities for deaf sign language users in countries such as Swaziland.