The encephalitic West Nile virus and its nonneuroinvasive variant, WN-25, were used to study the effect of macrophage depletion on viral invasion of the central nervous system. The in vivo elimination of macrophages was achieved by use of liposome-encapsulated drug dichloromethylene diphosphonate. Depletion of macrophages had an exacerbating effect on the course of the viral infection, exhibited by higher and extended viremia and accelerated development of encephalitis and death. Using a low dose of West Nile virus (5 PFU/mouse), an increase in mortality (from 50% to 100%) due to macrophage depletion was demonstrated. Furthermore, the attenuated noninvasive variant WN-25 showed high and prolonged viremia in the macrophage depleted mice (≈5 log 10 PFU/ml versus 2 in control mice), that allowed the penetration of the virus into the central nervous system. The mortality rate caused by the attenuated virus in the macrophage-depleted mice was 70–75%, as compared to complete survival in the control inoculated mice. These results indicate a significant role of macrophages in the non-specific immediate defence system of the organism in case of viral infection.