Calpactin I, a heterotetrameric, cytoskeletal protein complex composed of two copies of annexin II cross-linked by two copies of p11, an S100-like protein, binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments (GF) in a Ca(2+)-dependent manner, apparently without affecting GFAP polymerization under the present experimental conditions. Cosedimentation of calpactin I with GF, which occurs at micromolar free Ca2+ concentrations, is proportional to the concentrations of both calpactin I and GFAP and does not occur under conditions where GFAP assembly is maximally inhibited by, e.g., S100 protein. Annexin II also cosediments with GF and binds to GFAP, although to much smaller extents. Other annexins, such as annexins I, V, and VI, or p11 do not bind to either GF or GFAP. Calpactin I and S100 protein bind to different sites on GFAP, as investigated by fluorescence spectroscopy using acrylodan-labeled GFAP. Calpactin I and S100 protein might act, in the presence of Ca2+, in a concerted manner to determine the number and topography of GF in differentiating and/or mature glial cells.