Deoxyribonuclease I (DNase I) was purified from the semen of a 38-year-old male and then characterized. The catalytic properties of the purified enzyme closely resembled those of DNase I purified from the urine of this individual and the following other similarities were observed: molecular masses, iodoacetic acid inactivation kinetics, desialylated isoenzyme patterns. However, the behavior of the purified enzymes determined on several different lectin-affinity chromatography columns differed, which suggests that organ-specific glycosylation of DNase I occurs. Multiple forms of the purified seminal DNase I were demonstrated, each of which had a different pI value separated by isoelectric focusing, which is compatible with the reported existence of genetic polymorphism of seminal DNase I (Sawazaki et al., Forensic Sci Int 1992;57:39-44). Furthermore, enzymological and immunological comparisons of purified seminal and urinary and partially purified prostatic DNases I indicated that the prostate may be one of seminal enzyme source tissues.