Organotypic cultures of the spinal cord of the embryonic mouse were subjected to pulses of tritiated thymidine at various times between explanation and 42 days in vitro (DIV). Autoradiography was performed both on cultures fixed immediately at the end of the pulse and on cultures maintained in radioactive-free medium for various periods after the pulse. Quantitative light autoradiographic studies showed a single peak of glial cell proliferation at 9 DIV equivalent to that demonstrated in vivo. The growth rate of glial cells (related to time in culture) decreased along an exponential decay type curve. All these observations were statistically significant when tested against the corresponding null hypothesis. Ultrastructural autoradiography shows that at early stages of the culture, radial glial cells and immature glial cells divided and eventually gave rise to astrocytes and oligodendrocytes. During the period of maximal cell proliferation, tritiated thymidine was incorporated by differentiated astrocytes and ultrastructurally recognizable immature oligodendrocytes. Oligodendrocytes did not divide beyond the stage of active oligodendrocytes (the cells initiating myelination). They were capable of producing dark oligodendrocytes within a week following the last division. These observations emphasize the similarity of the proliferation during development in organotypic culture to that in vivo, modified by the trauma of explantation and the culture conditions.