The sensitivities for Chagas disease diagnosis of haemoculture, xenodiagnosis, and polymerase chain reaction (PCR) amplification of Trypanosoma cruzi kinetoplast deoxyribonucleic acid (DNA) were compared for 101 patients living in an endemic region who were serologically positive for T. cruzi. PCR gave 60 positive results (59.4%), while a haemoculture was positive in 26 cases (25.7%) and xenodiagnosis in 36 (35.6%). Four xenodiagnosis-positive but PCR-negative patients were examined in detail. The discrepancies were not due to inhibition of the PCR reactions, as the samples were used successfully to amplify a human sequence. Nor were they due to a variation in kinetoplast DNA sequences, as the kinetoplast DNA of the parasite strains isolated from these patients after xenodiagnosis gave rise to the expected product when amplified by the PCR. We concluded that no parasite was present in the 5 mL of blood used for PCR, while probably a single T. cruzi cell was present in the blood volume ingested by the insects during xenodiagnosis (about 3 mL). This suggests that the total blood quantity collected for the PCR may be important with patients with low parasitaemia.