Immunohistochemical techniques and antibodies to gamma-aminobutyric acid (GABA), parvalbumin, and cyclic guanosine monophosphate-dependent protein kinase were used to identify populations of cerebellar neurons in culture that exhibit morphological features and immunoreactivity characteristic of neuronal types present in the cortical region of the cerebellum in vivo. The cultures were examined at 3 culture ages: 6-9, 12-15 and greater than 15 days in vitro, reflecting early, intermediate and late periods in cerebellar development. Neurons identified as Purkinje neurons (PNs), granule cells or inhibitory interneurons (stellate, basket, Golgi and Lugaro cells) were present at all culture ages. The granule cells (GCs) and inhibitory interneurons (INs) were morphologically well developed at the youngest culture age studied; morphological features did not change dramatically during the culture period. In contrast, the PNs were morphologically immature at 6-9 DIV (DIV = days in vitro) and exhibited dramatic changes in morphological structure with culture age. Extracellular recordings from PNs. GCs and INs in living cultures revealed that all classes of neurons exhibited spontaneous activity, but that only a portion of the GCs and INs were spontaneously active. The spontaneously active GCs and INs exhibited variable patterns of activity and low firing rates (approximately 2-6 Hz) at all culture ages studied. At 6 DIV, PNs exhibited firing rates and patterns similar to that of the interneurons. At older culture ages, the firing rate and pattern of PNs was significantly different from the GCs and INs and was characterized by high frequency (greater than 10 Hz) spike activity usually in a regular pattern. All cerebellar neurons by excited by the transmitter glutamate (Glu). The Glu response in the GCs and INs consisted of a brief burst of single spikes; in PNs, the response to Glu was prolonged and multiphasic. These data indicate that the cerebellar GCs and INs express morphological, physiological and developmental properties that are significantly different from the PN.