Citrulline residues are detected in keratins and filaggrin in the cornified layers of mammalian epidermis. Such citrulline residues are formed by the enzymatic deimination of arginine residues by peptidylarginine deiminases (EC 3.5.3.15). Major deiminated keratins are derived from keratin K1. Two arginine residues identified as preferred deimination sites in mouse K1 are located in its V subdomains. To develop an immunochemical probe which recognizes the deiminated peptide sequence specifically, we enzymatically deiminated an undecapeptide corresponding to the deiminated peptide sequence identified in the V2 subdomain for immunizing rabbits. An IgG fraction obtained from the antiserum was affinity-purified using an immobilized peptide column. The affinity-purified IgG showed high specificity towards partially degraded keratin K1 obtained from the cornified layer of 3-day-old mouse epidermis. It also yielded intense signals of unidentified minor components localized in the cornified layers of late embryonic and early postnatal mouse epidermis. Comparative studies using different types of the enzymes suggested that peptidylarginine deiminase type I acted on the arginine residue in the V2 subdomain of keratin K1 more readily than peptidylarginine deiminase type II. The data are discussed in conjunction with possible factors influencing the specificity of the enzyme reaction.