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Detection of HIV proteins and/or nucleic acids is necessary for the diagnosis of perinatal HIV infection. Despite its low sensitivity, detection of p24 antigen in plasma is a simple and economic method for the diagnosis of HIV in exposed children. The aim of this study was to improve the sensitivity of detection of p24 using centrifugation of plasma. Forty-seven selected stored samples from 37 children (23 infected, 14 uninfected, median age of 137 days) were examined. Plasma samples (volume 0.3-1.5 ml) were defrosted, centrifuged at 23,500 x g at 4 degrees C for 60 min and determination of p24 was carried out in the resuspended pellet (0.12 ml). In 32 plasma samples from infected children, p24 was found originally in 6 (18.7%) and resulted positive in 24 (75%) pellets. When only one sample per child was considered, sensitivity was significantly higher in pellets, 3/23 uncentrifuged plasma samples and 15/23 pellets (McNemar Test, p<0.001). Specificity was 100%. The absorbance/cut-off ratio was always higher in the pellets from positive children (p=0.028). Plasma samples with volumes of 1 ml or more achieved a higher sensitivity (91.7% vs. 36.4%, p=0.009). Centrifugation of plasma samples prior to determination of p24 in pediatric patients resulted in a significant increase in sensitivity.