Explant outgrowths from human fetal brain were infected with 104 plaque forming units of the E variant of the encephalomyocarditis virus. Ultrastructurally, the majority of the cultured cells were astrocytes containing a moderate amount of glial fibrils. The earliest alterations at 44 hrs after infection of the culture consisted of dilatation of the rough endoplasmic reticulum (ER) and moderate enlargement of the mitochondria with increased density of their matrix. Twenty hours thereafter, increased amounts of the rough ER and of free ribosomes were observed in the infected cells. This was followed by aggregates of larger dense particles which developed into a parallel lattice-like pattern within the cytoplasm, presumably presenting viral particles, without obvious cytonecrosis. The present observations of the continuous development of the crystalloid formation of the virus within the cytoplasmic matrix as well as the mode of formation of the free ribosomes adjacent to the ER support the previous hypothesis that, after the uptake of the virus into the cytoplasmic matrix, it associates with the ER, where production of viral RNA polymerase and of viral RNA is initiated.