OBJECTIVE To examine the distribution of the collagen alpha 1(IV) chain and a novel collagen alpha(IV)-related chain in human ocular tissue. METHODS Two monoclonal antibodies, JK199 and M3F7, against the alpha 1(IV) chain, and one monoclonal antibody, JK132, against a novel alpha(IV)-related chain were used in the avidin biotin peroxidase complex procedure of immunohistochemical studies. In situ hybridization and reverse transcription polymerase chain reaction were used to examine the presence of alpha 1(IV) messenger RNA in corneal epithelium. RESULTS Our data indicate that monoclonal antibodies JK199 and M3F7 react with most ocular basement membranes, but not with those of corneal epithelium. Similarly, monoclonal antibody JK132 reacts with most basement membranes of ocular tissues, with the exception of the inner limiting membrane of neural retina, Bruch's membrane, and corneal epithelial basement membrane. To examine if the epitopes recognized by the monoclonal antibodies were masked in corneal epithelium, the tissue sections were subjected to limited enzyme digestion, that is, pepsin, hyaluronidase, trypsin, and pronase E, or chemical treatments such as 0.1 N NaOH or 6 M urea. Proteinase treatment removed the JK132 epitope from all ocular basement membranes examined. Despite the pretreatment, corneal epithelial basement membrane was not stained by any of the monoclonal antibodies. However, the alpha 1(IV) messenger RNA was detected in corneal epithelium by in situ hybridization and reverse transcription polymerase chain reaction. Western immunoblotting indicates the presence of the alpha 1(IV) and the novel alpha(IV)-related chain in the basal lamella of corneal epithelium. CONCLUSIONS The epitopes recognized by JK199, M3F7, and JK132 are masked in basement membrane of corneal epithelium. Based on the tissue distribution and partial amino acid sequences of peptides recognized by JK132, the novel alpha(IV)-related chain differs from other known alpha(IV) chains.