We describe a method that has allowed us to measure the synthesis, turnover and assembly of α- and β-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. For these studies, rat hippocampal cultures containing 74.5-83.0% neurons were established. B-27 (Gibco) supplement has been used to obtain an excellent long-term viability (up to 5 weeks) of hippocampal neurons in culture. For the synthesis, turnover, and assembly experiments the neurons were labeled with [ 3 5 S]methionine, and chased with 10-fold excess of cold methionine for the turnover experiments. The cells were then lysed and immunoprecipitated with α, β-erythroid, α, and β-nonerythroid spectrin antibodies. Immunoprecipitated [ 3 5 S]methionine-labeled spectrins of hippocampal neurons grown in vitro produced bands in 5% polyacrylamide minigels strong enough to be detected by the high sensitivity screens of a phosphorimager to generate graphs from which the synthesis or half-lives of α, β-erythroid, α, and β-nonerythroid spectrins were calculated. This method can be used to study the role of calpain, caspase-3, and the ubiquitin-proteasome system on the synthesis and turnover of erythroid and nonerythroid spectrins in resting and depolarized rat hippocampal neurons in culture.