Universal HIV testing of pregnant women in the United States is the key to prevention of mother-to-child transmission of HIV. Repeat testing in the third trimester and rapid HIV testing at labor and delivery are additional strategies to further reduce the rate of perinatal HIV transmission. Prevention of mother-to-child transmission of HIV is most effective when antiretroviral drugs are received by the mother during her pregnancy and continued through delivery and then administered to the infant after birth. Antiretroviral drugs are effective in reducing the risk of mother-to-child transmission of HIV even when prophylaxis is started for the infant soon after birth. New rapid testing methods allow identification of HIV-infected women or HIV-exposed infants in 20 to 60 minutes. The American Academy of Pediatrics recommends documented, routine HIV testing for all pregnant women in the United States after notifying the patient that testing will be performed, unless the patient declines HIV testing ("opt-out" consent or "right of refusal"). For women in labor with undocumented HIV-infection status during the current pregnancy, immediate maternal HIV testing with opt-out consent, using a rapid HIV antibody test, is recommended. Positive HIV antibody screening test results should be confirmed with immunofluorescent antibody or Western blot assay. For women with a positive rapid HIV antibody test result, antiretroviral prophylaxis should be administered promptly to the mother and newborn infant on the basis of the positive result of the rapid antibody test without waiting for results of confirmatory HIV testing. If the confirmatory test result is negative, then prophylaxis should be discontinued. For a newborn infant whose mother's HIV serostatus is unknown, the health care professional should perform rapid HIV antibody testing on the mother or on the newborn infant, with results reported to the health care professional no later than 12 hours after the infant's birth. If the rapid HIV antibody test result is positive, antiretroviral prophylaxis should be instituted as soon as possible after birth but certainly by 12 hours after delivery, pending completion of confirmatory HIV testing. The mother should be counseled not to breastfeed the infant. Assistance with immediate initiation of hand and pump expression to stimulate milk production should be offered to the mother, given the possibility that the confirmatory test result may be negative. If the confirmatory test result is negative, then prophylaxis should be stopped and breastfeeding may be initiated. If the confirmatory test result is positive, infants should receive antiretroviral prophylaxis for 6 weeks after birth, and the mother should not breastfeed the infant.