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Objective: In this study, "Pulsed-Field Gel Electrophoresis" (PFGE) protocol was optimized for use Providencia stuartii's typing in Molecular Microbiology Research and Application Laboratory, Public Health Agency of Turkey. Methods: This protocol compared with Gram-negative bacteria PFGE protocols, SpeI enzyme is suitable for this bacterium. Electrophoresis conditions should be revealed as 1. block: initial pulse duration 5 sec, ending pulse duration 40 sec, striking angle 120°, the current 6 V/cm2, temperature 14°C, time 19 hours; 2. block: initial pulse duration 35 sec, ending pulse duration 45 sec, striking angle 120°, the current 6 V/cm2, temperature 14°C, time 5 hours; TBE buffer pH=8.4. Phylogenetic analyzes were evaluated with Bionumerics software system (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium). Results and conclusion: This procedure is simple, reproducible and suitable for P. stuartii. It has the potential evaluating the outbreaks due to bacteria performed, and providing useful information about the prevalence of nosocomial infections. This optimized protocol is allowed different centers' PFGE results to compare with other laboratories results. [ABSTRACT FROM AUTHOR]