Medulloblastoma, a highly malignant pediatric tumor of the posterior fossa, demonstrates a marked propensity for leptomeningeal dissemination. Although the predominant site of relapse is the posterior fossa, the prevention of subarachnoid spread would be of significant therapeutic value. The established medulloblastoma cell lines D283 Med, D341 Med, D384 Med, D425 Med, D458 Med and Daoy have been investigated in in vitro adhesion assays for their capacity to bind to the predominant components of the leptomeningeal extracellular matrix: fibronectin, laminin and collagen IV. Growth on the reconstituted basement membrane matrix, Matrigel, was also assayed. Of the five neuronal phenotype DMed lines, all of which grow spontaneously as macrospheroids in standard fetal calf serum-containing tissue culture medium, only D425 Med and its sibline, D458 Med, derived from a subsequent sample from the same patient, displayed adherence to any of the substrata: approximately 20% of input D425 Med cells attached and exhibited cell spread and some extension (adhesion) on fibronectin. All other DMed lines failed to attach to these substrates. The glial phenotype cell line Daoy, which grows as an adherent monolayer under normal culture conditions, exhibited attachment, extension and growth on all substrata as did the glioma cell line U-251 MG and the neuroblastoma cell line SK-N-SH. The lack of attachment, and thus spread on components of the leptomeningeal extracellular matrix under in vitro assay conditions by 5/6 of the medulloblastoma cell lines investigated, is characteristic of neuronally differentiated cells, thus reinforcing the previously described neuronal phenotype of these lines. The readily demonstrated expression of N-CAM and L1 by all of the medulloblastoma cell lines suggests that the primary mode of leptomeningeal extension in vivo may be dependent on such other cell-cell and cell-substrate binding mechanisms.