Western blot was evaluated for the neonatal diagnosis of congenital toxoplasmosis based on a comparison of antibody profiles between serum samples obtained from the mother at delivery and from the neonate. Passively transferred antibodies can be distinguished from antibodies produced by the neonate, thus allowing early postdelivery diagnosis of congenital toxoplasmosis before the results of other tests are available. This method was developed at the Parasitology-Mycology laboratory of the Pitié-Salpêtrière Teaching Hospital, Paris, France, then evaluated in a retrospective study of 52 mother-infant pairs. The diagnosis of congenital toxoplasmosis was ruled out in 34 cases, confirmed in ten cases, and doubtful in 8 cases. Sensitivity was higher than with conventional serological tests. Antibody profile differences were found between mothers and affected infants; these differences usually involved IgGs (8 of 9 cases). Importantly, in two cases Western blot would have provided the diagnosis of congenital toxoplasmosis two months before the secondary elevation in IgM titers in one case and three weeks before the result of mouse placenta inoculation in another case. In conclusion, Western blot deserves to be used to complement established methods (serology and direct demonstration of the parasite by gene amplification, cell cultures, and mouse inoculations) as a means of rapidly (within 24 hours of receipt of the specimen) providing clinicians with information relevant to treatment decisions.