Quail embryos (embryonic days 2-2.5) with spontaneous neural tube defects (NTDs), along with age-matched normal embryos, were examined immunocytochemically for the extracellular matrix (ECM) molecules laminin, fibronectin, and chondroitin sulfate proteoglycan, the cell adhesion molecules (CAMs) E- and N-cadherin and neural CAM (NCAM), and the neural crest marker HNK-1. The embryos with NTDs were at the lower limit of the normal stage range and the affected region was about 25% shorter than in normal embryos. Open NTDs occurred in cervical and upper thoracic level, although often the ventral neural tube was morphologically normal. Widened, irregular but closed neural tubes (lower thoracic to sacral levels) showed disorganized mesenchyme-like cells centrally and often multiple lumens. Finger-like tabs projecting from the ectoderm over the neural tube also occurred at lower thoracic to sacral levels. In open NTDs, the E-cadherin-labeled epidermis was incomplete dorsally, and was continuous with the N-cadherin-labeled neural tissue, with a sharp demarcation between E- and N-cadherin-expressing regions, as in the early stages of normal primary neurulation. A sharp inverted peak of epidermis extended ventrally, closely applied to the side of the neural tissue. The intervening matrix labeled less intensely for chondroitin sulfate proteoglycan relative to laminin and fibronectin, in comparison to control embryos. In closed NTDs, the dorsal superficial cell layer (i.e., positionally epidermis) was not separated from the underlying neural tissue by a band of matrix as in control embryos. In addition, this layer expressed E-cadherin (as in normal embryos), but coexpressed N-cadherin and NCAM, which are not normally found here at this stage. This overlap region resembled the mid-dorsal tissue at earlier stages in normal secondary neurulation in the tail-bud. The tabs of tissue appeared to be localized hypertrophy of the epidermal and neural ectoderm, and also showed codistribution of E- and N-cadherin. In all these defects, matrix molecules occurred within (rather than around) the neural and epidermal epithelia. HNK-1-labeled neural crest cells were frequently absent in regions of NTDs, in contrast to control embryos. These results show that matrix and cell adhesion molecules are disturbed in spontaneous NTDs at the time of neurulation, and therefore could be involved in the generation of the defects by altering cell adhesion-dependent morphogenetic events.