Rotation-mediated aggregating cell cultures of mechanically dissociated fetal rat brains (15-16 days) were morphologically characterized at 4, 19, 26 and 40 days in vitro. The dissociated cells coalesced into spherical aggregates which increased in diameter from 340 micrometer at 4 days to 430 micrometer at 40 days. Cells within the aggregates developed from an undifferentiated state at 4 days to a population of morphologically mature neurons, astrocytes and oligodendrocytes (26 days in vitro) before degenerating. Synaptic contacts and myelinated axons appeared as the cells differentiated. Neurons tended to occur in clusters that were located in central regions of the aggregates, whereas astrocytes were more concentrated in the periphery. Synapses and myelinated axons were more abundant in central portions of the aggregates. The amount of myelin formed within the aggregates was less than in organotypic cultures or in vivo. These results show that the morphological differentiation of mechanically dissociated aggregates resembles the development of rat CNS in vitro. The ease with which large amounts of aggregates can be prepared provides an in vitro system which can be analyzed biochemically without the use of micro-methods. This advantage is particularly useful for multidisciplinary investigations in developmental neurobiology.