Definitive diagnosis of leptospirosis has traditionally depended upon the isolation of leptospires from clinical specimens or the demonstration of seroconversion in paired acute and convalescent serum samples. Both of these approaches require expertise not routinely available in clinical laboratories and usually result in delayed diagnosis. Conventional PCR assays have been developed, but all have limitations which have restricted their widespread use. In order to overcome these limitations, a real-time PCR assay was developed using a 423 bp target on the lipL32 gene, which is conserved among pathogenic serovars of LEPTOSPIRA: Reactions were monitored by SYBR green fluorescence and melting curve analysis. Representative serovars from 16 species of Leptospira and over 40 species of other bacteria and fungi were tested. Positive results were obtained with all pathogenic leptospiral serovars, with the exception of Leptospira fainei serovar Hurstbridge. The analytical sensitivity of this assay was 3 genome equivalents per reaction; approximately 10 genome equivalents were detectable in human urine. Leptospiral DNA was amplified from blood containing EDTA or citrate anticoagulants, but heparin, sodium polyanetholesulfonate and saponin were inhibitory. The assay successfully detected leptospiral DNA from serum and urine samples of patients with leptospirosis. This assay has the potential to facilitate rapid, sensitive diagnosis of acute leptospirosis.