OBJECTIVES: To elucidate the epidemiology of widespread ciprofloxacin resistance in our Veterans Affairs medical center using whole cell DNA analysis. DESIGN: In vitro study of ciprofloxacin resistant and susceptible Enterobacteriaceae isolated during the course of a clinical epidemiologic study of quinolone resistance. SETTING: Veterans Affairs Medical Center with acute care and long-term care divisions. RESULTS: We examined 40 ciprofloxacin-resistant strains of Serratia marcescens, Proteus mirabilis, and Providencia stuartii using restriction endonuclease analysis of whole cell DNA and compared them with concomitantly isolated ciprofloxacin sensitive strains. We sought to determine whether resistant strains were identical to susceptible strains, indicating in vivo emergence of resistant strains from susceptible strains, and whether resistant strains were shared among patients. All 26 ciprofloxacin-resistant S marcescens isolates shared a single ecoRI restriction pattern. Multiple patterns were seen in the ciprofloxacin-susceptible S marcescens isolates; however, several isolates had a pattern matching that of the resistant isolates. Similar results were seen among the P mirabilis isolates. Three different ecoRI patterns were found among the ciprofloxacin-resistant P stuartii isolates; none matched those found among the susceptible isolates. The frequency of spontaneous emergence of ciprofloxacin resistance in susceptible S marcescens strains with restriction pattern matching that of the resistant strains was significantly higher than that of nonmatching strains. CONCLUSIONS: Ciprofloxacin-resistant strains of Enterobacteriaceae became widespread within a short period of time. Resistant strains of S marcescens and P mirabilis arose from endemic susceptible strains. The resistant strain of S marcescens appeared to arise from a susceptible strain with a relatively high frequency of spontaneous ciprofloxacin resistance.