Validation studies that meet TWGDAM (The Working Group on DNA Analysis Methods) and CAC (California Association of Criminalists) guidelines for RFLP (restriction fragment length polymorphism) analysis were performed with the DNA probe EFD52 (D17S26). These studies demonstrate that the probe EFD52 is suitable for forensic casework. No unexpected DNA banding patterns were obtained from controlled studies examining various tissues, sample consistency over many gels, mixtures of body fluids, various substrates, various contaminants and non-human DNA sources. Of all the animal DNAs tested, only one higher primate yielded a single band to EFD52 hybridization. The sensitivity of EFD52 was shown to be comparable to that of other forensic probes. Population frequency distribution tables were prepared from over 4000 alleles and two-locus studies were conducted on nine forensically useful probes. Black, White, Hispanic and Lumbee Indian populations were found to be in Hardy-Weinberg and linkage equilibrium. Comparisons between victim blood standards and epithelial fractions of mixed stains from sexual assault cases were used to demonstrate the robustness of the EFD52 probe in forensic casework.