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Congenital toxoplasmosis is rarely identified by routine clinical examination. The aim of this study was to estimate the incidence of the disease in the region of Ribeirão Preto, south-eastern Brazil. A definitive diagnosis was made on the basis of the persistence of anti-Toxoplasma IgG antibodies beyond 1 year of age. Blood samples obtained from 15162 neonates and adsorbed onto filter paper were tested for anti-Toxoplasma IgM antibodies. Fifteen samples gave positive results. A definitive diagnosis was confirmed in five of the 13 infants (38·5%) who completed follow-up. These five infants presented with serum IgM and/or IgA antibodies, and clinical abnormalities. Disease incidence was estimated to be 3·3/10000 (95% CI 1·0–7·7), indicating the need for preventive measures. Neonatal screening is feasible, but screening tests with a better performance are required; positive screening results must be carefully confirmed.