Flat mounts of human corneal endothelial cells (HCECs) were examined immunohistochemically by using a wide assortment of monoclonal antibodies against the five classes of intermediate filaments (IFs) and actin and myosin. HCECs showed uniform immunostaining with monoclonal antibodies against the 40-kD (CK 19) and 45-kD (CK 18) cytokeratin (CK). Only part of the endothelial cells reacted with monoclonal antibodies against the 52-kD (CK 8) and 54-kD (CK 7) cytokeratin polypeptides and with monoclonal antibodies against vimentin. Monoclonal antibodies against the low- and middle-molecular-mass neurofilament proteins produced positive staining of all HCECs. No positivity was obtained with antibodies against desmin or glial fibrillary acidic protein. In addition, positive immunostaining with monoclonal antibodies against actin and slow myosin demonstrate that these proteins form part of the cytoskeleton of HCECs. The results of this study show that immunostaining of flat cell preparations is very useful for studies on HCECs. HCECs display an unusual combination of cytokeratin IFs and neurofilaments, together with vimentin, and are heterogeneous with respect to their IF makeup. These findings are discussed in relation to the presumed origin of HCECs.