Objective: To compare the effect of treatment decisions guided by phenotypic resistance testing (PRT) or standard of care (SOC) on short-term virological response. Design: A prospective, randomized, controlled clinical trial conducted in 25 university and private practice centers in the United States. Participants: A 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. Interventions: Randomization was to antiretroviral therapy guided by PRT or SOC. Main outcome measures: The 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). Results: At 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). Conclusion: Antiretroviral treatment guided prospectively by PRT led to the increased use of 'active' antiretroviral agents and was associated with a significantly better virological response. © 2002 Lippincott Williams & Wilkins.