A clinical feature of Ross River virus disease (RRVD) is the periodic relapse of symptoms months after the initial onset of disease. The underlying mechanisms responsible for this relapse have not been determined. In a long-term (148 days) in vitro study of persistently infected murine macrophages we established that RRV infection periodically fell to undetectable biological levels that required genetic detection. However, the virus concentration spontaneously relapsed to biologically detectable levels that corresponded with enhanced viral mRNA expression, cellular detachment, and cytopathic effect. By altering the cell culture conditions we found that relapse could also be induced. We propose that the periodic relapse of symptoms in RRVD may be associated with spontaneous or stress-induced increases in RRV within persistently infected macrophages. This study also established that RRV enhanced macrophage phagocytic activity and dysregulated the immunoregulatory molecules CD80, IFN-gamma, and TNF-alpha that may facilitate persistence of RRV and avoidance of immune responses.