Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemorr~gi~-deriv~L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-11) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and 6 2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-11, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.