Aims To study the association between number and positions of mutations with MICs of fluoroquinolone non-susceptible Haemophilus influenzae. Methods and Results More than 40% of 48 H. influenzae isolated from nursing home residents were not susceptible to fluoroquinolone. Amino acid changes in the quinolone resistance determining regions, and correlation with MICs and inhibition zone diameters were analysed. All isolates with reduced susceptibility to fluoroquinolones (MIC >= 0 center dot 125 mu g ml(-1)) had at least one mutation in gyrA at position 84 and were resistant to nalidixic acid. Compared to isolates with reduced susceptibility, resistant isolates were associated with mutations in gyrA at positions 88 and 134, and in parC at position 88 (P < 0 center dot 001). Inhibition zone diameter for nalidixic acid disk >= 23 mm may detect susceptible isolates. Conclusions Reduced susceptibility to fluoroquinolones was associated with mutations at position 84 in gyrA. A further increase in fluoroquinolone MIC was associated with mutations in gyrA at positions 88 and 134, and parC at position 88. Significance and Impact of the Study Due to limited resistant H. influenzae strains, prior studies on association between positions of mutations and fluoroquinolone MICs were inconclusive. The comparison of mutations between isolates with susceptibility, reduced susceptibility and high resistance supported the importance of the present study.