Hemorrhagic fever with renal syndrome (HFRS) has been serologically confirmed in Slovenia during the last seven years. There is evidence that three hantaviruses (Hantaan, Puumala, and a newly described form termed Dobrava) circulate simultaneously in this area. Recently, a hantavirus was isolated from the urine and brain tissue of a fatal case of HFRS. Positive immunofluorescent reactions with reference human sera and monoclonal antibodies were first recognized after the second cell culture passage. Extensive cross-reactivity between our isolate and prototype Hantaan virus, strain 76-118, and Hantaan-like isolates from the former Yugoslavia, Fojnica and Plitvice, was revealed by enzyme-linked immunosorbent assay with specific rat antisera. The reaction pattern of the isolate was similar to the prototype Hantaan virus by indirect immunofluorescent assay with a panel of monoclonal antibodies. Furthermore, the specificity of the isolates was confirmed by analysis of polymerase chain reaction products of this virus with five restriction endonucleases. This appears to be the first isolation of a strain of prototype Hantaan virus from a fatal case of HFRS in Europe.