A developmentally regulated, cytoskeletal-associated protein was identified and partially characterized using a monoclonal antibody developed for this study. Based on the distinctive fibrous pattern of distribution of this antigen in the cytoskeletons of cultured corneal fibroblasts, and a characteristic reorganization of these fibers into perinuclear whorls in response to colchicine treatment, this protein was found to be associated with the intermediate filaments (vimentin filaments). Chronological distribution of this intermediate filament-associated protein (IFAP) and vimentin during fetal development of the cornea in rabbit was analysed immunohistochemically. During an early stage of corneal development (day 13 of gestation), both this IFAP and vimentin were present in the stromal cells in the presumptive corneal region. The IFAP was also present in the surface epithelium. At day 17 and 21, the corneal endothelial layer was developed and contained both the IFAP and vimentin. However, in the corneal stromal cells, the concentration of IFAP (based on the densities of the immunostaining reaction) progressively decreased during the later stages of fetal development (day 24 to day 28), while relative concentrations of vimentin were not reduced significantly. In the corneal stromal cells in the adult rabbit, the IFAP was not detectable while vimentin was still present. For further characterization of this protein, extracts of cultured corneal fibroblasts and fetal corneas were analysed by SDS-PAGE followed by an immunotransblot technique. These analyses indicated that the IFAP was a polypeptide with a Mrof 130k. Therefore, this protein was not vimentin (Mr55–58k). The presence of this IFAP in the fetal corneas and stromal cells and its absence in the quiescent stromal cells in the adult cornea indicated that this unique IFAP is developmentally regulated in corneal stromal cells, and its association with vimentin filaments may be important during the active state of corneal stromal cells.