This pilot study established that unlinked anonymous testing of dried blood spots routinely collected on Guthrie cards for neonatal screening is a feasible method for monitoring HIV prevalence in women at the time of delivery. The method was sensitive, specific, and less expensive than more conventional ELISAs. 114,515 dried blood spots taken from cards collected in three Thames regions were tested for antibody to HIV-1. 28 samples were confirmed to be antibody positive by western blot (seroprevalence 0.24 per 1000). Unlinked anonymous screening of newborn babies should be extended to monitor the spread of HIV infection in the heterosexual population and to target preventive strategies and provision of health care.