Macrorestriction analysis of genomic DNA followed by pulsed-field gel electrophoresis (REA-PFGE) has become the 'gold standard' for molecular typing (10). In recent years, some alternative techniques have been successfully applied. However, there is still a need for a new method which may appear to be less complex, cheaper, and have a power of discrimination at least similar to that of REA-PFGE. Recently, we showed the performance and convenience of a novel assay, based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting), for its potential usefulness in epidemiological investigations. In this study, ADSRRS-fingerprinting method was optimized at each stage of the procedure. The modified method, named ADSRRS-fingerprinting plus, differs from the original technique in several points. The optimized method considerably shortens the time of experiment, offers good discriminatory power and also demonstrates excellent reproducibility. Next, ADSRRS-fingerprinting plus was validated. These experiments proved that the method is specific, selective and suitable for intended purpose and also confirmed its reliability, reproducibility and simplicity in the interpretation of the results. Optimized and validated procedure of ADSRRS-fingerprinting plus was used in the development of a diagnostic kit (ADSRRS-KIT) for strain differentiation of bacteria below the species level, using a simple set of ready-to-use reagents and standard equipment.