The immunocytochemical visualization of neuron-specific enolase, which is a marker protein for differentiated neurons, was applied to follow the differentiation of preoptic and septal neurons in dissociated cultures. From 4 to 24 days in vitro, the relative numbers of stained neurons were counted and the staining intensity of individual neurons determined by absorbency measurements using a television-based densitometer. Whereas few stained cells could be observed at 4 DIV, 80% of the neurons were neuron-specific enolase-positive at 13 days in vitro. This value remained constant up to 24 days in vitro. The density of the immunoreaction product increased dramatically from 13 to 17 days in vitro and was still higher at 24 days in vitro. The glial and ependymal cells of the carpet, as well as neuroblasts, remained unstained. Comparison with morphological observations and immunocytochemical demonstration of neuronal peptides made earlier shows that expression of neuron-specific enolase closely parallels neuronal differentiation. These observations indicate that cultures derived from preoptic and septal neurons represent a viable model system for the study of neuronal maturation in vitro.