To determine the role of pregnancy on Trypanosoma cruzi parasitemia, a matched cohort study was carried out in a rural Bolivian community comparing parasite rates in gravidae, puerperae, and non-pregnant infected women. A selection of 67 chronically infected women, who delivered between March 2004 and May 2005, were initially evaluated during the third trimester of pregnancy and again after delivery. They were matched for age, parity, and location with 104 seropositive non-pregnant women, who likewise had submitted blood for microscopic examination for T. cruzi parasites in June 2005. Seroreactive pregnant women had a higher rate of T. cruzi parasitemia (14.9%) than matched non-pregnant infected women (2.9%; P = 0.004). After delivery, parasitemia significantly decreased during puerperium (1.5%) compared with the period of pregnancy (14.9%; P = 0.03). This study showed an increase of parasite loads in maternal peripheral blood, during the third trimester, and a significant decline after delivery.