For obtaining a deeper insight into the properties of a newly characterized member of the protein 4.1 family, brain 4.1, the cellular and subcellular localization was investigated in the cerebellar cortex of adult and postnatally developing rats. Fluorescent immunohistochemical observations showed that brain 4.1 localized predominantly to glomeruli in the granular layer and throughout the molecular layer in adult rat cerebellar cortex. Analysis of subcellular localization of brain 4.1 by immuno-electron microscopy further demonstrated that presynaptic terminals of mossy fibers and parallel fibers, cytoplasm of granule cells and cytoplasm and/or processes of glial cells contained brain 4.1 while postsynaptic regions of the dendrites of granule cells and Purkinje cells, axons and myelin sheaths did not. Thus, one of the major subcellular destination of brain 4.1 was presynaptic terminal in the cerebellum. This was further supported by the fact that the immunostaining pattern of brain 4.1 in the cerebellum changed in a similar way to that of a synaptic terminal marker, synaptophysin during the postnatal development. Immunoblot analysis also demonstrated that contents of brain 4.1 isoforms varied in parallel with the changes of the immunostaining pattern. Biochemical analysis confirmed the presence of brain 4.1 at synaptic terminals, but there was no obvious correlation between each isoform and its subcellular localization. These results suggested that brain 4.1 is involved in the formation and maintenance of synapse as a membrane skeletal component at presynaptic terminals in the cerebellum.