question: Abstract Mouse hepatitis virus (MHV) infection of the L-2 subline of mouse fibroblasts results in acute infection characterized by extensive cell fusion. In contrast, infection of the LM-K subline leads to virus persistence with reduced cell fusion. We undertook studies designed to elucidate the role of host cell membrane lipid composition and the cytoskeleton in modulating the fusion process and the resultant effect(s) on virus persistence. MHV-induced cell fusion proceeded normally in cells treated with cytoskeleton-disrupting drugs, cytochalasin B and colchicine. Modification of cell membrane fatty acid composition by supplementation of LM-K cells with arachidonic (C-20:4) or palmitic (C-16:0) acids had little effect on the extent of MHV-induced cell fusion or on virus replication. However, supplementation of both cell types with cholesterol (resulting in increased membrane cholesterol/fatty acid ratio) resulted in marked enhancement of virus-mediated cell fusion. The increase in cell membrane cholesterol did not enhance internalization of MHV suggesting that cholesterol primarily modulates a later event. This suggestion was confirmed by demonstrating cholesterol-enhancement of fusion in a contact fusion assay. Cholesterol-supplemented L-2 cells were less productive for virus replication than unsupplemented cells, in agreement with our previous observations that MHV replication is compromised by extensive cytopathic effect. Although cholesterol-supplemented LM-K cells showed increased susceptibility to MHV-mediated cell fusion, the extent of such susceptibility did not approach that observed in L-2 cells. Also, the property of LM-K cells to support MHV persistence was not abolished by cholesterol supplementation. Thus membrane fusion resistance and MHV persistence are modulated but not alleviated by cell membrane cholesterol content.