Objective: The testing of neonatal blood specimens dried on filter paper for maternal HIV antibodies, using an enzyme immunoassay (EIA) with confirmation of repeatedly reactive specimens by immunoblot (IB), was first described in 1987. It has been used to conduct large, unlinked, anonymous HIV seroprevalence surveys for surveillance of HIV in child-bearing women in several countries. We directly assessed the sensitivity and specificity of this combination of tests to detect maternal HIV antibodies. Setting: Serum samples obtained from mothers delivering at a major hospital in Kinshasa, Zaire were screened for HIV antibody using the rapid assay HIVCHEK. Design: Plasma from HIVCHEK-positive women and age-matched negative controls were tested by enzyme-linked immunosorbent assay (ELISA); repeatedly reactive specimens were confirmed by Western blot (WB). Two days after delivery, whole blood was obtained from each newborn by heel-stick, dried on filter paper, and tested by EIA. Repeatedly reactive specimens were confirmed by IB. Main outcome measure: The serologic status of neonatal filter-paper specimens was compared with that of corresponding maternal plasma. Results: The testing of neonatal filter-paper specimens using EIA, with confirmatory testing of repeatedly reactive specimens using IB, was 100.0% sensitive [of the 192 ELISA-positive and WB-positive maternal plasma specimens, 192 of the corresponding newborn filter-paper specimens were EIA-positive and IB-positive; 95% confidence interval (CD, 98.1–100]. The detection of maternal HIV antibodies was 99.6% specific using this combination of tests (of the 281 ELISA-negative or ELISA-positive but WB-negative maternal plasma samples, 280 of the corresponding newborn filter-paper specimens were EIA-negative or EIA-positive but IB-negative; 95% Cl, 98.0–100). Conclusions: Maternal HIV antibodies can be detected accurately by testing neonatal blood dried on filter paper, using EIA with confirmation of repeatedly reactive specimens by IB. This approach can facilitate the determination of HIV seroprevalence in child-bearing women in countries with neonatal screening programs, or where serum or plasma is difficult to obtain.