Abstract We have passaged the prototype strain of Murray Valley encephalitis virus in SW13 (human) cells, sequenced the E and M genes, and examined the virulence of the passaged virus for 21-day-old mice following intracranial and intraperitoneal inoculation. Six independent passage series were carried out: four in the presence of mouse hyperimmune ascitic fluid and two without antibody. Changes were observed in the E protein deduced amino acid sequence for each of the six 10th passage stocks sequenced. Eleven changes were observed in total for the six stocks sequenced; these were at residues 117, 118, 390, 423, and 460. Nine of the changes were nonconservative. Five of the six passaged variants were altered at Asp 390 which is part of an Arg-Gly-Asp (RGD) sequence. This change resulted from adaptation to SW13 cells rather than from antibody selection. The RGD sequence (and residue 423) falls within a region which is highly conserved between flaviviruses and is strongly hydrophilic. All five variants which were altered at Asp 390 were attenuated in 21-day-old mice following i.p. inoculation. We propose that the domain of E encompassing the RGD sequence is an important determinant of flavivirus pathogenicity.