Formation of the inner ear from the otic placode differs from invagination of other cup-shaped organ primordia. Activation of the actin cytoskeleton seems to play a limited role because precocious invagination does not occur upon treatment with activators of a contractile event and cannot be prevented by inhibitors. In this study, the possibility that invagination is mediated by changes in the surrounding mesenchyme was tested by treating embryos with agents which interfere with the integrity of extracellular matrix. Enzymes degrading hyaluronate and/or chondroitin sulfate were microinjected into the otic region prior to folding. Synthesis of chondroitin sulfate proteoglycan was inhibited by microinjection of β-xyloside. All treatment inhibited otic pit formation by interfering with fold formation within the placode. Immunocytochemical procedures showed depletion of the appropriate extracellular matrix components for a short time period after enzyme treatments and for up to 24 hr after β-xyloside injection. Invagination of the otic primordium is concluded to be controlled in part by anchorage of the epithelium to adjacent structures and possibly by expansion of the mesenchymal extracellular matrix. © 1995 Wiley-Liss, Inc.