The first aim of this study was to determine the prevalence of congenital toxoplasmosis in newborn infants treated by the public health system in Porto Alegre, a city in southern Brazil, using neonatal screening for Toxoplasma gondii-specific IgM. The second aim was to investigate whether the cases detected by this approach could have been identified by the prenatal screening for antibodies to T. gondii that was performed in the same population. A fluorometric assay was used to analyse T. gondii-specific IgM in filter paper specimens obtained from newborn infants for routine screening for metabolic diseases. When the specific IgM was positive, serum samples from the infant and the mother were requested for confirmatory serological testing, and the infant underwent clinical examination. Among 10 000 infants screened for T. gondii-specific IgM, seven filter paper samples were positive, and congenital toxoplasmosis was confirmed in six patients. The prevalence of IgM specific for T. gondii was 6/10 000 [95% CI 2/10 000, 13/10 000]. One infected infant had already been identified in the maternity ward before birth, three had been identified by maternal serology at delivery, and two infants with congenital toxoplasmosis were identified solely through neonatal screening. Although four mothers of the patients with congenital toxoplasmosis received prenatal care, and three mothers had one or two serological tests for T. gondii-specific antibodies (one at first trimester, one at first and second trimesters, and the other at second and third trimesters), they were not identified during pregnancy as infected. Neonatal screening identified cases of infection not detected by obtaining only one or two serum samples from pregnant women for T. gondii serology, mainly when infection was acquired and transmitted in late pregnancy. Maternal serology at delivery and neonatal screening were especially useful in the identification of infants with congenital toxoplasmosis when the mother did not receive regular prenatal serological testing or prenatal care.