The association of several phosphoproteins with the synaptosomal plasma membrane (SPM) was investigated by phosphorylating SPM fractions from neonatal rat brain in the presence of Ca2' and then exposing these to a variety of agents. Extraction of the major acidic phosphoproteins, GAP-43, pp40, and ppS0, was assessed by two-dimensional gel electrophoresis and fluorography. All three proteins were best extracted from the membrane by high pH and by guanidme hydrochloride. GAP43 was not extracted in the presence of either low-or high-ionic-strength buffers, reducing agents, or chelating agents; ppS0 and pp40, however, showed a significant extraction even under low-ionic-strength conditions. Partition experiments with Triton X-114 revealed an amphiphilic behavior for GAP43 and a strong affinity for hydrophobic environments for ppS0 and pp40. None of the phosphoproteins was released from the membrane by the use of a phosphatidylinositol-spe- cific phwpholipase C. The extraction properties of GAP-43, ppS0, and pp40 are similar to those of known extrinsic membrane proteins and therefore suggest that these phosphoproteins are peripheral rather than integral to the membrane compartment.