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SUMMARY: In situ DNA hybridization using biotin-labelled leptospiral DNA was performed on clinical specimens to investigate its usefulness as a technique for the identification of Leptospira interrogans. The applicability of this test in blood, urine and liver smears was demonstrated. In situ DNA hybridization can be completed in only 4 h and it combines the advantage of visualization of the leptospiral morphology with the specificity of the hybridization reaction. No cross-hybridization was observed with other bacteria. This study shows that hybridization in situ can be simple to perform and may contribute to a rapid diagnosis.