A sequential study of the early stage of development of ethylnitrosourea (ENU)-induced glioma in the rat was performed by electron microscopy and [3H]thymidine histoautoradiography. Hyperplasia, the earliest neoplastic change that was detectable morphologically, consisted of a few or several immature oligodendroglia-like cells which were connected with one another or with preexisting neural tissue by junctional apparatus, and showed no reactive changes in the astrocytes or microglia. The labelling index of hyperplastic cells was 2.6%. Foci of early neoplastic proliferation (ENP) showed mild destructive changes in the neighboring neural tissue, and their major constituent cells had characteristics of immature oligodendroglias. The labelling index of cells showing ENP was 3.3%. The intercellular spaces exhibited slight enlargement with accumulation of extracellular matrix and a decrease in the number of junctional apparatus on the neoplastic cells. Microtumors showed apparent destruction of the preexisting neural tissue to form a tumor mass with an increase in the extracellular matrix. Constituent cells of the microtumors were similar to those of the ENP, although reactive astrocytes and microglias occurred more frequently. The labelling index was 9.6% in the central area of microtumors and 5.3% in the peripheral area. These findings suggest that in the initial or very early stages of glial cell neoplastic proliferation, it is necessary for the neoplastic cells to maintain contact with the neurons for metabolic purposes, and that after losing contact, these cells can proliferate autonomously with the accumulation of extracellular matrix.