Sustained viral suppression with antiretroviral therapy improves clinical outcomes for HIV-infected individuals. Study M05-730 evaluated the long-term antiviral activity, safety, tolerability, emergence of resistance, and compliance with once-daily (QD) versus twice-daily (BID) lopinavir/ritonavir (LPV/r) combination therapy in treatment-naïve, HIV-1-infected subjects through 96 weeks. Antiretroviral-naïve subjects with HIV-1 RNA levels >1000 copies/ml were randomized to LPV/r QD (N = 333) or BID (N = 331) with tenofovir DF and emtricitabine. Through 96 weeks, 77 subjects from each group discontinued prematurely; adverse or HIV-related events contributed to discontinuation of 36 subjects overall, with no significant differences between treatment groups. At 96 weeks, 216 QD subjects (64.9%) and 229 BID subjects (69.2%) had HIV-1 RNA <50 copies/ml (p = 0.249) by intent-to-treat analysis. Evaluation of the time to virologic failure indicated that 85.0% and 80.7% of QD and BID subjects, respectively, maintained virologic suppression through 96 weeks (p = 0.638). QD subjects demonstrated greater adherence levels. There were no significant differences in virologic response when subjects were analyzed according to baseline disease state. Emergence of postbaseline resistance mutations occurred at similar low rates in each dosing group. Diarrhea was the most common moderate-to-severe drug-related adverse event reported; the most common Grade 3+ laboratory abnormalities were elevations of total cholesterol and triglycerides, occurring with similar incidence regardless of LPV/r dosing frequency. QD dosing of LPV/r was associated with similar durability of viral suppression and low rates of genotypic resistance and treatment-limiting adverse events as compared with BID dosing in treatment-naïve subjects through 96 weeks of treatment.