OBJECTIVES: To develop a polymerase chain reaction (PCR) method to detect Haemophilus ducreyi DNA in cultured isolates and clinical material. METHODS: Primers specific to the H ducreyi 16s rRNA gene were synthesised. PCR conditions were optimised and products were verified by restriction endonuclease digestion and agarose gel electrophoresis. RESULTS: The method was able to detect all 28 H ducreyi strains tested; specificity was demonstrated using lysates of 12 related organisms. Applied to clinical samples from genital ulcer swabs obtained in Harare, Zimbabwe, H ducreyi DNA was detected in repeated assays in 35 clinical samples. CONCLUSION: PCR amplification using primers from the 16s rRNA gene may be a useful alternative to culture for the detection of H ducreyi and the diagnosis of chancroid.