We have confirmed for the first time, both biochemically and immunologically, the existence of deoxyribonuclease I (DNase I) in the human tears. Isoelectric focusing of tear samples on polyacrylamide gels (pH 3.5-5), followed by dried agarose film overlay detection, was used to determine the phenotypes of tear DNase I. Because of its high degree of sensitivity and its high band resolution, this detection method allowed determination of DNase I types from tear samples of about 5 μL. Pretreatment of tear samples with the enzyme sialidase was essential for typing in order to markedly enhance the sensitivity and simplify the isozyme pattern. DNase I types in all tear samples were consistently related to types found in corresponding blood and urine samples. DNase I typing may, therefore, provide a novel discriminating characteristic in the genetic, clinical and forensic examination of tears.