Background: Lopinavir/ritonavir (LPV/r) monotherapy is used in selected virologically suppressed HIV-infected patients. Some would prefer a once-daily (OD) dose instead of the usual twice-daily dose to favour adherence. However, trough concentrations of the drug in blood and particularly in cerebrospinal fluid (CSF) may not be adequate to maintain viral suppression. Methods: Prospective, open-label pilot study to evaluate the efficacy and safety of LPV/r monotherapy OD. HIV-1-infected patients, virologically suppressed for at least 6 months were enrolled. HIV viral load (VL) was determined at baseline and at weeks 4, 8, 12, 16, 24, 36 and 48. Lumbar puncture was performed in a subgroup of patients to evaluate CSF VL and CSF LPV concentrations. Results: A total of 21 patients were included. At week 48, 85.7% (n=18) showed viral suppression (VL<40 copies/ml). Two patients had viral failure (9.5%) and a third was withdrawn from the study because of gastrointestinal symptoms. Nine patients were enrolled in the substudy. CSF VL was <40 copies/ml in all cases. Median (range) LPV concentration was 9.78 ng/ml (1.93-78.3) in CSF and 1,970 (154-16,700) ng/ml in plasma; the CSF/plasma ratio was 0.004 (0.001-0.186). Conclusions: In this small pilot study, LPV/r monotherapy OD maintained plasma HIV RNA suppression at 48 weeks in most patients, with no cases of CSF viral escape. However, CSF LPV concentrations were close to the 50% inhibitory concentration threshold in several patients; hence, this intervention should be avoided in patients with advanced immune suppression and/or those individuals presenting with significant comorbidities such as hepatitis C coinfection.