Introduction.Leptospirosis, caused by spirochetes of the genusLeptospira, is present in the Koprivnica-Križevci County area, Croatia. Clinical manifestation can range from asymptomatic, short-term mild, non-specific febrile disease, to severe forms with high mortality rates.Aim.The aim of the study was to valuate culture in front of microscopic agglutination test (MAT) for diagnosis of infection, and to evaluate clinical and laboratory features of the disease. Moreover, we want to characterizeLeptospirastrains involved in infection in Koprivnica-Križevci County, Croatia.Methods.We included 68 patients with clinical presentation consistent with leptospirosis collected in a 5-year period (2000–2004). Clinical samples (blood, urine and cerebrospinal fluid, CSF) were inoculated in Kolthoff’s medium; species of isolatedLeptospirastrains was determined with Tm of real-time PCR, serogroup/serovar with MAT and NotI-RFLP analysis. Demonstration of specific antibodies in patients’ sera was done using microscopic agglutination test.Results.Leptospirawas isolated from the blood of 14/51(27.5 %) patients and the most often identified serogroup/serovar was Icterohaemorrhagiae (8/10, 80%) followed by Grippotyphosa (10%). Regarding to species level, 8/10 isolated belonged toL. interrogans sensu strictoand one toL. kirschnerispecies. MAT was carried out on 51 patients with suspected leptospirosis, and was positive in 11/51(21.5 %) patients. Most of our patients presented with moderate severe symptoms, were hospitalized from August to October, and were infected mainly during the work or recreation in our county. The frequency of particular clinical features and pathological laboratory findings correlated with the severity of the clinical condition.Conclusions.Leptospirosis can be confirmed microbiologically, culture and MAT contributed almost equally to the diagnosis of infection. Serovar Icterohaemorrhagiae was found as the dominant one, andL. interrogans sensu strictoas dominant species in our county. Epidemiological data shown that leptospirosis occurs seasonally, affects the rural population, and most commonly is presented with moderate severe clinical course.