Levels of viral burden were compared across risk group and gender populations among 485 human immunodeficiency virus type 1 (HIV-1)-infected participants consisting of 190 male injection drug users (IDUs), 92 female IDUs, and 203 homosexual men. Viral burden was quantified by a microculture technique to determine cell-associated infectious units per 10⁶ peripheral blood mononuclear cells (IUPM) and by reverse transcriptase PCR (Amplicor) to determine plasma HIV RNA levels. Adjusting for CD4⁺ cell count, females had a lower infectious HIV load than all males combined (0.33 log₁₀ lower; P = 0.004), and homosexual men had a 0.29 log₁₀ higher infectious viral load than all IDUs combined (P = 0.001). For HIV RNA levels, females had lower levels than males (0.19 log₁₀ lower; P = 0.04), but no differences were observed by risk group. After controlling for percent CD4⁺ cells, no differences were found by risk group for either assay, but females still had a 0.25 log₁₀ lower infectious viral load than males (P = 0.04) and a viral RNA load similar to that of males (P = 0.25). The correlation between infectious viral load and HIV RNA load was 0.58 overall, which did not differ by gender or risk group. Our data suggest that differences in viral load may exist by gender and that any differences observed by risk group are driven predominantly by gender or percent CD4⁺ cell differences. These data also confirm a moderate correlation between cell-associated infectious viral load and plasma HIV RNA load, which appears to be similar by gender and across risk groups.