The mesenchyme of the elevating mesencephalic neural folds of the mouse is composed primarily of mesenchymal cells embedded in an hyaluronate-rich extracellular matrix. In this study we provide evidence that hyaluronate and mesenchymal expansion may play a role in neural fold elevation and closure. Spatial and temporal patterns of mesenchymal cell and hyaluronate distribution were analyzed during neural fold elevation and closure using the computer-assisted method of smoothed spatial averaging and established methods of image processing. Degree of fold elevation and fold shape changes were analyzed using standard morphometric measures. The results of these analyses defined five distinct stages in mesencephalic neural fold elevation and closure. Mesenchymal cells and hyaluronate were found in a non-random distribution within the neural fold and showed distinct patterns of distribution which could be correlated with specific stages in neural fold elevation. The results of these analyses suggested that the elevation of the mesencephalic neural folds is produced by the expansion of an hyaluronate-rich extracellular matrix in the central mesenchyme which under the direction of surrounding tissues pushes the folds mediad towards the dorsal midline.