An inexpensive and technically less-demanding methodology to quantify HIV-1 viral load would be of great value for resource-limited settings, where the nucleic-acid amplification technique (NAAT) is impractical and/or resource-prohibitive. In this study, an HIV-1 reverse-transcriptase enzyme-activity assay (ExaVir Load assay, version 1) was compared with the gold standard RT-PCR assay, Roche HIV-1 Amplicor Monitor, version 1.5. A total of 121 plasma specimens were used for the evaluation. ExaVir Load had a sensitivity of 97 % and a specificity of 71 % in identifying specimens with <400 copies ml(-1) in the Roche RT-PCR assay as being less than the detection limit of the assay (5500 copies ml(-1)). The mean difference (95 % limits of agreement) between Roche RT-PCR and ExaVir Load was -0.23 (-1.59 to 1.13) log(10)(copies ml(-1)) by Bland-Altman analysis. Significant negative correlations were seen between CD4(+) T-cell counts and the ExaVir Load assay (r=-0.32, P<0.05), and between CD4(+) T-cell counts and the Roche RT-PCR (r=-0.38, P<0.01). The present study with HIV-1 showed a strong correlation between the ExaVir Load assay and the RT-PCR assay. Hence, the ExaVir Load assay could be considered for use in resource-limited settings as an alternative viral-load assay to the standard NAAT-based assay after further evaluation with prospective specimens.