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Objectives To evaluate the correlations of the combination of undetectable HIV-DNA (<10 copies/106 peripheral blood mononuclear cells) and HIV-RNA (<1 copy/mL of plasma) levels and a CD4 cell count of >500 cells/mm3 (defined as the treatment goal) in a group of 420 antiretroviral treatment (ART) responder patients. Methods A cross-sectional, open-label, multicentre trial was conducted in a cohort of 420 HIV-infected ART-treated subjects with viral loads persistently <50 copies/mL for a median observation time of 28.8 months. HIV-DNA and residual viraemia values and demographic, virological and immunological data were collected for each subject. Results Undetectable HIV-DNA was found in 16.6% (70/420) of patients and was significantly correlated with undetectable (<1 copy/mL) plasma viraemia (P = 0.0001). Higher CD4 cell count nadir (P < 0.001), a lower HIV-RNA viraemia at the start of treatment (P = 0.0016) and nevirapine use (P < 0.001) were correlated with an undetectable value of HIV-RNA. Twenty-six out of 420 patients (6.2%) reached the treatment goal. In multivariate analysis, higher nadir CD4 cell count (OR 3.86, 95% CI 1.47–10.16, P = 0.006), the duration of therapy (OR 1.07, 95% CI 1.02–1.12, P = 0.004) and the use of nevirapine (OR 2.59, 95% CI 1.07–6.28, P = 0.034) were independently related to this condition. Conclusions Only 6.2% of ART-responder patients presented the combination of three laboratory markers that identified them as full responders. These results indicate the high variability of the ART-responding population and lead us to suggest caution in the selection of patients for possible simplification regimens [ABSTRACT FROM AUTHOR]