Using the polymerase chain reaction (PCR) assay, we developed a rapid diagnosis method for Salmonella typhi infection in blood specimens from patients with typhoid fever. Primers were designed from the flagellin gene sequence, which would give an amplification product of 367 base pairs. In this study, the specificity of the assay, with no amplification, was seen for the other Salmonella strains with the flagellin gene, and not for non- Salmonella bacteria. For the sensitivity test, the protocol described allowed the detection of two to three copies of the Salmonella typhi genome, as determined by serial dilution of genomic DNA from Salmonella typhi. With the PCR technique, genomic DNA of Salmonella typhi was detected in 46 of 73 blood samples collected from patients with clinically suspected typhoid fever who had fever within 3 days of admission to the General Hospital, Makassar, South Sulawesi, Indonesia, and who had had no prior antibiotic treatment. The PCR results (63% positive cases) were compared with those of blood culture (13.7% positive cases) and the Widal test (35.6% positive cases), using the same samples from each of the 73 patients admitted to the General Hospital in Makassar. The time taken for PCR analysis of each sample was less than 12 h, compared with 3 to 5 days for blood or clot culture. The PCR with one pair of primers can be used as a novel, rapid diagnostic method for typhoid fever, particularly when results of standard culture assays are negative.