Using broad-spectrum primers, we have amplified, cloned, and sequenced a 620-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp65) from the heart valve of a patient with Whipple's endocarditis. The deduced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific seminested PCR. Seventeen patients shown to be positive by 16S ribosomal DNA (rDNA) PCR and/or internal transcribed spacer (ITS) PCR were also positive by hsp65 PCR. All 33 control specimens from patients without Whipple's disease and negative for "T. whippelii" by both seminested 16S rDNA and ITS PCR remained negative. All amplicons digested with XhoI revealed an identical restriction pattern. Eight of the 17 hsp65 amplicons representing all three previously described ITS types were sequenced. Three of the amplicons showed slight differences, but none of the mutations detected affected the amino acid sequence of the corresponding protein. We conclude that the hsp65 gene is a suitable target for the specific detection of "T. whippelii." Its product represents a putative antigen for a future serodiagnostic assay.