Within the central nervous system, apolipoprotein E (apoE) synthesis is increased in response to nerve injury, a finding that may reflect a role for apoE in neuronal remodeling. Recent studies show that apoE3 promotes and apoE4 inhibits neurite outgrowth in cultured neuronal cells. Interestingly, these isoform-specific effects are observed only when apoE is presented to cells in the presence of an exogenous lipid source such as rabbit beta-very low density lipoprotein (beta-VLDL), making it difficult to discern the biologically active form of apoE or to understand the role of the lipid source. In the present study we tested whether a cell-derived lipidated form of apoE can alter neurite outgrowth in the absence of beta-VLDL by constructing Neuro-2a cell lines expressing high levels of apoE. Our results showed that endogenous apoE3 stimulated neurite outgrowth, whereas the endogenous apoE4 isoform was neutral. Furthermore, beta-VLDL antagonized the stimulatory effects of the endogenous apoE3. Characterization of the secreted apoE3 indicated that the neurite outgrowth-stimulating activity could be recovered from culture medium with an anti-apoE immunoaffinity column and was present in a poorly lipidated particle with a density between 1.19 and 1.26 g/ml. These results indicated that the biological activity of apoE3 in stimulating neurite outgrowth was inherent in the cell-derived apoE particle and was not dependent on either (a) an interaction of apoE3 with an artificial lipid source or (b) independent actions of apoE3 and beta-VLDL.