The effect on central myelin of Actinomycin D, an RNA--and, secondarily, a protein-synthesis inhibitor, has been studied by light and electron microscopy. The intracranial injection of this drug produced an extensive status spongiosus of the white matter in the cerebrum, cerebellum, brain stem and optic nerve within 48 h. The status spongiosus was due to vacuole formation within the myelin sheath and to enlargement of the extracellular space. Three types of vacuoles were observed: (a) the most common varieties formed between the inner tongue and the remainder of the myelin sheath; (b) a second variety formed by enlargement of the periaxonal space with separation of the axon from its myelin sheath, and (c) a less common type of vacuolization was due to splitting of the myelin lamellae at the interperiod line to form large intramyelinic vacuoles. Myelinic vacuoles were preceded by nuclear and cytoplasmic changes in oligodendrocytes, which included nucleolar segregation, disaggregation, and diminution in number of ribosomes. These changes were similar to those previously reported in a variety of cells exposed to Actinomycin D. It is suggested that myelin vacuoles result secondarily from the Actinomycin D inhibitory effect on oligodendroglial RNA--and protein-synthesis, rather than from a direct effect of this drug on the myelin sheath.