The middle sized (M) RNA segment of California group viruses (Bunyaviridae) is a major determinant of virulence for mice. This has been demonstrated by intraperitoneal (ip), or intracerebral (ic), inoculation of 4-week-old mice with prototype or reassortant bunyaviruses, the latter obtained from mixed infection of BHK-21 cells with temperature sensitive (ts) mutants of La Crosse (LAC), snowshoe hare (SSR), and Tahyna (TAR) viruses. Reassortant viruses were not more virulent than wild-type progenitor viruses. All reassortants, and wild-type cloned viruses having the LAC, or SSH, M RNA segment, killed mice following ip inoculation; reassortant and wild-type viruses having the TAH M RNA did not kill 4-week-old mice after ip inoculation. In addition, reassortant virus with the TAH M RNA killed mice after ic inoculation more rapidly than did viruses having the LAC M RNA segment, irrespective of the parental origin of the small (S), or large (L), viral RNA species. The correlation of the M RNA segment with virulence implies that G1 and/or G2 determine major murine virulence properties, perhaps through attachment which may influence tropism. These experiments are an initial step in understanding the mechanism of virulence for mice, and eventually for man. They could also lead to the rational design of live attenuated bunyaviruses as vaccines for man and domestic animals.