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Neurologic and hematologic abnormalities are common in HIV-infected children and may be related to concomitant deficiencies in serum B12 and folate, which are highly prevalent in HIV-infected adults. We sought to determine the prevalence of B12 and folate deficiencies in HIV-infected children in the United States.Cross-sectional information on demographics, folate and B12 levels, hematological parameters, concurrent CD4%, HIV-viral load and antiretroviral regimens were abstracted from the medical records of 103 vertically infected children followed in an outpatient pediatric HIV clinic in the Bronx, during 2001–2002.Mean age was 10 years (±4.4 years), 46% were male, 53% African–American and 46% Hispanic. Nineteen percent had significant immunologic suppression and 18 children had AIDS. All were receiving combination antiretroviral therapy and 66% were on a protease inhibitor-based regimen. Sixteen were taking cotrimoxazole prophylaxis. None were taking multivitamins or manifested clinical evidence of gastrointestinal malabsorption. All patients had serum folate or B12 levels within or above the normal range. Children with elevated B12 were significantly more likely to be younger (P = 0.0002) and have higher mean folate levels (P = 0.0004) compared with children with normal serum B12. In a multivariate logistic regression analysis, factors independently associated with elevated levels of vitamin B12 included: elevated serum folate [odds ratio (OR): 3.2; P = 0.01], nonnucleoside reverse transcriptase inhibitor use (OR: 0.38; P = 0.05) and female sex (OR: 0.67; P = 0.42)Folate and B12 deficiencies are uncommon in HIV-infected children in the United States, suggesting that routine supplementation with B12 and folate is not indicated without confirmation of micronutrient deficiency.