Background: A progressive frequency of resistance to fluorquinolones is observed among Gram-negative bacilli. Aim: To investigate the mechanism of resistance to fluoroquinolones mediated by mutations affecting gyrA and gyrB genes in strains of Gram negative bacilli isolated from Chilean hospitals. Material and method: Minimal inhibitory concentration of fluoroquinolones was determined in 91 randomly selected nalidixic acid-resistant strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, isolated from hospitals of 12 Chilean cities. Quinolone resistance determining region (QRDR) was amplified by PCR and mutations were determined by restriction fragment length polymorphism (RFLP) and DNA sequencing. Results: Strains with mutation in codon 83 of gyrA showed decreased susceptibility to ciprofloxacin with MICs ranging from 0.25 to 1024 mu g/ml. The sequencing of PCR products for gyrA indicated amino acid changes in the QRDR region. One strain of E. coli presented a double mutation in codon 83 Ser to Leu as well as in codon 8 7 Asp to Asn. In strains of K pneumoniae, however, the change of codon 83 was Ser to Tyr, in A. baumannii was Ser to Len and in R aeruginosa was Thr to Ile. No strains with mutations affecting gyrB were found. Conclusions: Mutations in codon 83 of gyrA is a frequent genetic event involved in the mechanism leading to decreased susceptibility to fluoroquinolone in strains of Gram-negative bacilli.