Calymmatobacterium granulomatis is the aetiological agent of granuloma inguinale - a chronic granulomatous genital infection - and is morphologically similar to members of the genus Klebsiella. This study determined the 16S rRNA gene sequence of C. granulomatis and the taxonomic position of the organism in relation to the genus Klebsiella. Genomic DNA was extracted from C. granulomatis-infected monocytes and from frozen and formalin-fixed paraffin wax-embedded tissue biopsy specimens from patients with histologically proven granuloma inguinale. The 16S rDNA was amplified by PCR with broad range oligonucleotide primers. The amplified DNA fragments were cloned into pMOS vector, digested with Bam HI and Pst1 restriction endonucleases, hybridised with a gram-negative bacterial probe (DL04), sequenced in both directions by the automated ALF DNA sequencer, verified on an ABI Prism 377 automated sequencer and analysed with DNASIS and MEGA software packages. Sequence analysis revealed DNA homology of 99% in C. granulomatis from the different sources, supporting the belief that the bacteria in the culture and the biopsy specimens belonged to the same species, although there was some diversity within the species. Phylogenetically, the strains were closely related to the genera Klebsiella and Enterobacter with similarities of 95% and 94% respectively. C. granulomatis is a unique species, distinct from other related organisms belonging to the gamma subclass of Proteobacteria.