We tested 617 dried blood spots (DBS) from human immunodeficiency virus-exposed infants from five countries using an ultrasensitive p24 antigen assay (Up24). The sensitivity was 94.4% (67/71) and the specificity was 100% (431/431) for infants with DBS specimens <or=20 months old; DBS older than 30 months demonstrated only 72.2% sensitivity (39/54) (P < 0.001) but displayed 100% specificity (61/61).