Cultured cerebellar granule neurons develop their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, initially extending a single process, followed by the extension of a second process from the opposite pole of the cell, both of which develop into axons to generate a bipolar morphology. A mature morphology is attained following the outgrowth of multiple, short dendrites [Powell et al., 1997: J. Neurobiol. 32:223-236]. To determine the relationship between the localization of the Golgi apparatus, the site of microtubule nucleation (the centrosome), and the sites of initial and secondary axonal extension, the intracellular positioning of the Golgi and centrosome was observed during the differentiation of postnatal mouse granule neurons in vitro. The Golgi was labeled using the fluorescent lipid analogue, C5-DMB-Ceramide, or by indirect immunofluorescence using antibodies against the Golgi resident protein, alpha-mannosidase II. At 1-2 days in vitro (DIV), the Golgi was positioned at the base of the initial process in 99% of unipolar cells observed. By 3 DIV, many cells began the transition to a bipolar morphology by extending a short neurite from the pole of the cell opposite to the initial process. The Golgi was observed at this site of secondary outgrowth in 92% of these "transitional" cells, suggesting that the Golgi was repositioned from the base of the initial process to the site of secondary neurite outgrowth. As the second process elongated and the cells proceeded to the bipolar stage of development, or at later stages when distinct axonal and somatodendritic domains had been established, the Golgi was not consistently positioned at the base of either axons or dendrites, and was most often found at sites on the plasma membrane from which no processes originated. To determine the location of the centrosome in relation to the Golgi during development, granule neurons were labeled with antibodies against gamma-tubulin and optically sectioned using confocal microscopy. The centrosome was consistently co-localized with the Golgi during all stages of differentiation, and also appeared to be repositioned to the base of the newly emerging axon during the transition from a unipolar to a bipolar morphology. These findings indicate that during the early stages of granule cell axonal outgrowth, the Golgi-centrosome is positioned at the base of the initial axon and is then repositioned to the base of the newly emerging secondary axon. Such an intracellular reorientation of these organelles may be important in maintaining the characteristic developmental pattern of granule neurons by establishing the polarized microtubule network and the directed flow of membranous vesicles required for initial axonal elaboration.