Objectives. To determine the prevalence of antibodies against Trypanosoma cruzi in puerperal women and to assess possible congenital transmission of Chagas' disease in the department of Arequipa, Peru, where the disease is endemic. Methods. Women who had given birth between December 2001 and July 2002 in three hospitals (two urban and one rural) and four health centers (three rural and one urban) of the department of Arequipa, Peru, were studied. The serological study included screening all the puerperal women in order to detect antibodies against T. cruzi through indirect immunofluorescence (IIF), with confirmatory testing done with enzyme-linked immunosorbent assay (ELISA) testing and the titration of immunoglobulin G (IgG) antibodies by IIP. IIP tests to screen for immunoglobulin M (IgM) antibodies were done with the seropositive women and their newborns, and infection was evaluated through xenodiagnosis (evaluated at 30 and 60 days) and the direct micromethod of Freilij et al. The results were analyzed in terms of the presence of the vector and of cases of Chagas' disease in the places where the puerperal women had been born and where they were living. Two neonatologists clinically evaluated the newborns in order to detect abnormalities and signs of congenital Chagas' disease. Results. The overall prevalence of Chagas' disease in the 3 000 puerperal women studied was 0.73%. Prevalence was highest in two health centers located in rural areas (2.2% in El Pedregal and 4.1% in La Joya) (P = 0.018). The disease was associated with previous direct contact with the vector (P < 0.05) and with having been born in an area considered endemic (P < 0.01). Four (20%) of the 20 seropositive puerperal women were also positive by xenodiagnosis. However, none of the women was aware of her infectious carrier state, and none showed the characteristic symptoms or signs of acute or chronic Chagas' disease. IgM antibodies were not detected in any of the puerperal women. One neonate (whose mother did not have evidence of parasitemia) presented an IgM titer of 1/8, but in later controls neither IgM nor IgG antibodies were detected. Parasites were not detected in the blood of the neonates by either of the two testing methods used. Of the 20 neonates evaluated, one presented microcephaly and hepatosplenomegaly; although the child had specific IgG antibodies against T. cruzi at birth, the antibodies were not present at the age of two months. The growth and development of the other 19 newborns were normal. Conclusions. The prevalence of Chagas' disease in puerperal women of the department of Arequipa, Peru, is low. No cases of intrauterine congenital transmission were found. We recommend carrying out studies on prenatal detection that evaluate more mothers and in which women who give birth at home also participate.