Most of the experiments were performed with a human DNA standard purchased from Promega. DNA was isolated from a number of samples to validate the assay. The samples were blood spots from seven DNA databank samples; three samples from control blood spotted on denim; control blood placed on eight surfaces; sets of two blood spots placed either in the dark, in sunlight, or at 37 degrees C in the dark for 3 months; male and female fractions from four sexual assault cases and one case standard; three samples from a proficiency test; six swabs from various surfaces; swabs of three fingerprints; saliva from three envelope seals; and a blank from a DNA extraction. DNA was isolated by using an organic extraction method as modified in Akane et al. Animal DNA was isolated from samples of blood on paper or cloth or buccal swabs of pets of laboratory personnel, using the organic extraction method. Real-time PCR for the Alu assay was performed in a Corbett Research Rotorgene. Various initial experiments changed annealing and/or extension times and temperatures to optimize the assay. A melt curve was also performed after the assay to check for specificity of the reaction. The quantiblot kit was used for the slot blot detection of human DNA. The AMPFlSTR COfiler kit was used for STR analyses. The real-time Alu assay was found to have many advantages over the current slot blot assay as well as over the Alu plate reader assay. The assay is cost effective and is fast, requiring approximately 0.5 hours of setup time and 72 minutes of PCR amplification. The quantitation results are immediately calculated by the real-time instrument software and can be printed out. Also, the dynamic range is larger than the slot blot assay. The variation of assay conditions does not have any major effect on the assay, suggesting it may be robust over the variations between instruments or laboratories. 6 figures, 5 tables, and 21 references