Based on the hypothesis that the attachment of neuroectodermal cells to thrombospondin-1 (TSP-1) may affect tumor spread and play a role in the anti- tumor effects of retinoic acid, we investigated the expression of TSP-1 in these cells in situ and the effect of retinoic acid on the morphology of TSP- 1-adherent neuroblastoma (SK-N-SH) and malignant astrocytoma (U-251MG) cells in vitro. TSP-1-adherent SK-N-SH cells demonstrated process outgrowth, with further neuronal differentiation after retinoic acid treatment, consistent with the in situ studies showing that TSP-1 expression occurs in a differentiation-specific manner in neuroblastic tumors. TSP-1-adherent U- 251MG cells failed to spread; however, after retinoic acid treatment the cells demonstrated broad lamellipodia containing radial actin fibers and organization of integrins α3β1 and α5β1 in clusters in lamellipodia and filopodia. The attachment of both SK-N-SH and U-251MG cells to TSP-1 was found to be mediated by heparan sulfate proteoglycans, integrins, and the CLESH-1 adhesion domain first identified in CD36. Heparin and heparitinase treatment inhibited TSP-1 attachment. Integrins α3β1 and α5β1 mediated TSP-1 attachment of SK-N-SH cells, and integrins α3β1, α5β1 and αvβ3 mediated TSP-1 attachment of U-251MG cells. Attachment was dependent on the RGD sequence which is located in the carboxy-terminus of TSP-1. Treatment with a pharmacologic dosage of retinoic acid altered the TSP-1 cell adhesion mechanism in both cell lines in that neither heparin nor micromolar concentrations of the RGD peptide inhibited attachment; after treatment, attachment was inhibited by the CSVTCG peptide located in the type I repeat domain of TSP-1 and a recombinant adhesion domain (CLESH-1) from CD36. Expression of CD36 was found in the retinoic acid-treated U-251MG cells. These data indicate that neuroectodermally derived cells utilize several mechanisms to attach to TSP-1, and these are differentially modulated by treatment with retinoic acid. These data also suggest that the CSVTCG sequence of TSP-1 modulates or directs cytoskeletal organization in neuroblastoma and astrocytoma cells.