Previous studies demonstrating the presence of collagen II in the basilar membrane have used a biochemical approach or have used immunohistochemistry at the light microscopic level. In this investigation both the presence and arrangement of collagen II were demonstrated at the ultrastructural level using pre- and post-embedding immunoelectron microscopy. Labeling was dependent on the development of protocols to expose epitopes while maintaining identifiable ultrastructure. Both positive and negative controls indicate that the labeling was specific for collagen II. Collagen II was detected in the fibrous sheet of the pars tecta and in the two fibrous layers of the pars pectinata. It was detected in situ and on isolated individual 10-12 nm fibrils. The presence of collagen II in all the fibrous layers of the basilar membrane places constraints on the biomechanical properties of this important structure.