In preparation for the application of restriction fragment length polymorphism (RFLP) analysis methods to casework, validation studies were performed at the Indianapolis-Marion County Forensic Services Agency (IMCFSA). They encompassed the following areas: standard specimens, sample consistency, reproducibility, mixed specimens, environmental insults, minimum sample requirements, non-probative casework, repetitive sizing accuracy, precision and match criteria analysis, and population studies including K562 data analysis. All studies yielded results as expected, were consistent with those previously reported by other laboratories, and formed a strong foundation on which to base future casework analysis.