The organization of microfilaments using NBD-phallacidin and cell adhesion to substratum by surface reflection interference microscopy was examined during differentiation of astrocytes in colony cultures and correlated with motile behaviour of cells. Disaggregated cells from neopallium of 12-day-old or newborn DBA/1J mouse embryos were used to establish colonies and astrocyte precursor cells at various stages of differentiation along the astrocyte lineage were examined after 3 days, 1, 2 and 4 weeks in culture. The earliest astrocyte precursor cells, the glioblasts, are stationary and form epithelial-type colonies which adhere to the substratum primarily around the edge where large microfilament bundles are found. Bundles of microfilaments are also present around the apical ends of closely packed cells. As the epithelial cells start to separate and transform into flat proastroblasts, adherens-type junctions which have a zig-zag appearance and are associated with microfilaments form between adjacent cells. In the highly motile astroblasts these junctional regions break down into multiple smaller regions where the separated cells remain in contact through fine processes. The astroblasts also have stress fibres, focal contacts with substratum, foci from which microfilament bundles radiate and a complex pattern of fine, circumferentially oriented bundles of microfilaments. This elaborate organization of microfilaments disappears as the motile astroblasts differentiate into stationary fibrous astrocytes that have little polymerized actin and lack focal contacts. These results show that stationary astrocyte precursor cells in vitro go through a highly motile stage having a characteristic distribution of microfilaments and focal contacts before becoming stationary again. We consider that the motile stage could correspond to the stage in vivo when astrocyte precursor cells migrate from the ventricular and subventricular regions to take up position in different parts of the developing brain.