We describe the use of deoxyribonuclease I (DNase I) polymorphism for individualization of semen in body fluid stain mixtures, as a means of providing new and more useful information to practicing forensic biologists as a genetic marker. We have already reported that human DNase I isozyme patterns from different subjects are classifiable into ten groups. Isoelectric focusing of DNase I isozymes on polyacrylamide gel (IEF-PAGE, pH 3.5 to 5) was accomplished using a 0.5 mm thick gel. Pretreatment of semen samples with neuraminidase enhanced the isozyme band resolution and sensitivity. Activity detection using the dried agarose film overlay (DAFO) procedure was reliable, sensitive and simple, with high resolution, and the phenotypes of DNase I were determined in semen stains of about 0.3 microL stored at room temperature for up to a year in most of the samples tested. The DNase I types in semen stains were correlated with the types found in the corresponding blood and urine samples, although most of the vaginal fluid samples had no typable DNase I activity. This is considerably advantageous for seminal individualization from body fluid mixture stains in criminal cases. An evaluation of DNase I typing by IEF-PAGE and DAFO was also performed on casework samples submitted to our laboratory, and the results showed that DNase I was expected to be one of the most useful individualization marker of semen in practical application.