We modified and evaluated a RIA for serum and used the modified RIA to measure zidovudine in dried blood spot specimens (DBSs) routinely collected for newborn screening and tested anonymously for maternally acquired HIV antibodies in the national HIV Seroprevalence Survey Among Childbearing Women. DBS calibration and quality-control materials were used to adapt the serum assay to the DBS matrix. The assay had a limit of detection of 24 micrograms/L serum and was used to measure zidovudine from both whole DBSs and the eluate remaining after HIV antibody screening. We initiated a pilot study to investigate the assay's performance and assess its potential to determine the implementation of the US Public Health Service recommendations that HIV-infected pregnant women and newborns receive zidovudine treatment to reduce the risk of perinatal HIV transmission.