ObjectiveTo compare the effect of treatment decisions guided by phenotypic resistance testing (PRT) or standard of care (SOC) on short-term virological response.DesignA prospective, randomized, controlled clinical trial conducted in 25 university and private practice centers in the United States.ParticipantsA total of 272 subjects who failed to achieve or maintain virological suppression (HIV-1-RNA plasma level > 2000 copies/ml) with previous exposure to two or more nucleoside reverse transcriptase inhibitors and one protease inhibitor.InterventionsRandomization was to antiretroviral therapy guided by PRT or SOC.Main outcome measuresThe percentage of subjects with HIV-1-RNA plasma levels less than 400 copies/ml at week 16 (primary); change from baseline in HIV-1-RNA plasma levels and number of ‘active’ (less than fourfold resistance) antiretroviral agents used (secondary).ResultsAt week 16, using intent-to-treat (ITT) analysis, a greater proportion of subjects had HIV-1-RNA levels less than 400 copies/ml in the PRT than in the SOC arm (P= 0.036, ITT observed;P= 0.079, ITT missing equals failure). An ITT observed analysis showed that subjects in the PRT arm had a significantly greater median reduction in HIV-1-RNA levels from baseline than the SOC arm (P= 0.005 for 400 copies/ml;P= 0.049 for 50 copies/ml assay detection limit). Significantly more subjects in the PRT arm were treated with two or more ‘active’ antiretroviral agents than in the SOC arm (P= 0.003).ConclusionAntiretroviral treatment guided prospectively by PRT led to the increased use of ‘active’ antiretroviral agents and was associated with a significantly better virological response.