Our aim was the detection of possible deficiencies of folate and cobalamin by the measurement of plasma total homocysteine (tHcy) in 69 human immunodeficiency virus (HIV) -infected children on antiretroviral treatment. We studied the relationship of these vitamins and methionine with tHcy values. Plasma tHcy was determined by high-performance liquid chromatography with fluorescence detection, folate and cobalamin by competitive protein-binding chemiluminescence, and methionine by ion exchange chromatography. Significant differences were observed between tHcy concentrations in the HIV-infected patients and the reference values for children of similar ages (P < 0.0001). Folate values were significantly lower in HIV-infected children compared with our reference paediatric population (P < 0.0001), but cobalamin concentrations were similar between patients and reference values. A significantly negative correlation was found between tHcy and folate (r = - 0.596; P < 0.0001), and a significantly positive correlation between folate and the methionine : tHcy ratio (r = 0.501; P < 0.0001). Plasma tHcy was significantly higher (P = 0.008), while folate values and methionine : tHcy ratios were significantly lower (P = 0.007 and P = 0.042), in patients on protease inhibitor treatment than in patients on other antiretroviral therapies. The hyperhomocysteinaemia and low methionine : tHcy ratios observed in our group of HIV-infected children are probably a consequence of the low folate values, which interfere in the remethylation of homocysteine to methionine. Patients on protease inhibitor treatment showed significantly higher plasma tHcy concentrations, and lower folate values and methionine : tHcy ratios, compared with patients on other antiretroviral therapies. Hyperhomocysteinaemia is associated with the risk of premature stroke, which may have adverse consequences in the evolution of disease.