Microtubule-associated protein 1B (MAP1B) is expressed mainly in the brain during early development and plays important roles in the regulation of microtubule dynamics which is essential to neurite outgrowth and elongation. Recent studies report, however, that MAP1B persists in some areas of mature brain where it may serve functions other than microtubule-binding, in some cases possibly as a transmembrane protein. To understand the entire aspect of MAP1B function, we investigated the expression and subcellular localization of MAP1B during the course of synaptogenesis in cultured rat cortical neurons. Major part of synaptogenesis in this system took place between 3 and 17 days in vitro as monitored by Synapsin I expression. After surface-biotinylation of intact cells, subcellular fractionation was carried out using streptoavidin-conjugated magnetic beads to yield three fractions: plasma membrane fraction with attached membrane skeleton, cytoskeletal fraction, and soluble fraction. The amount of total MAP1B as well as the proportion of cytoskeletal MAP1B was kept constant between 7 and 21 days. MAP1B in the plasma membrane fraction increased progressively at the expense of soluble MAP1B, reaching 50% of total at 21 days in vitro. A small but reproducible proportion (0.35%) of MAP1B was also detected as a biotinylated transmembrane protein which increased with synaptogenesis. There was a concomitant increase in plasma membrane-associated actin, indicating the development of actin-based membrane skeleton. It is thus concluded that MAP1B has another important role in the maturation of neurites through establishment of the membrane skeleton.