Aims: The aim of the present study was to rapidly optimize enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification systems for fingerprinting rat's intestinal microflora. Methods and Results: Orthogonal array design and statistic analysis methods were attempted to rapidly optimize ERIC-PCR reaction system for fingerprinting intestinal microflora. The results showed that variations of the four factors (Mg2+, dNTP, primer and HotstarTaq polymerase concentrations) changed the fingerprinting patterns significantly. The order of effects of those factors on fingerprinting patterns was primers (F = 274.000, P = 0.000), Hotstar Taq polymerase (F = 197.000, P = 0.001), Mg2+ (F = 181.000, P = 0.001) and dNTP (F = 27.000, P = 0.011). The optimal ERIC-PCR condition was containing 200 mu mol l(-1) dNTP, 2.5 mmol l(-1) Mg2+, 0.4 mu mol l(-1) primer, 1 U HotstarTaq DNA polymerase namely 25 mu l reaction system, which is proved to be a simple, fast and reliable method suitable for fingerprinting rat's intestinal microflora. Conclusion: The results suggest that Mg2+, dNTP, primer and HotstarTaq polymerase concentrations play important roles on ERIC-PCR fingerprinting patterns. Orthogonal array design is a considerable method to optimize ERIC-PCR reaction system for its rapidness, simplicity, potential to investigate mutual effects of parameters. Significance and the Impact of the Study: It is the first report on optimization of ERIC-PCR amplification systems for fingerprinting intestinal microflora using orthogonal array design or statistic analysis methods and systematically observing the effects of variables of reaction conditions.