Studies looking at the frequency of human herpesvirus-8 (HHV-8) among Iranian blood donors have produced conflicting results. The aim of this study was to investigate the prevalence of HHV-8 DNA by using polymerase chain reaction methods among 168 healthy individuals, 60 intravenous drug users and 100 HIV-infected patients from Iran. The prevalence of HHV-8 was significantly higher among intravenous drug users (13.3%) compared with the general population (3.6%; P=0.017). The HHV-8 genome was mostly detected among intravenous drug users who displayed high-risk sexual behaviours. Moreover, the HHV-8 genome was also detected in 8% of HIV-infected patients. The present study findings support the likelihood that the transmission of HHV-8 is via a sexual route in the Iranian population.