In 1999, a cluster of gonococcal isolates exhibiting high Minimal Inhibitory Concentrations (MICs), to azithromycin (2.0-4.0 mg/l) were identified in Kansas City, MO. Isolates were characterized by auxotype/serovar class, lipoprotein (Lip) subtyping and sequencing of the mtrR gene, which has been implicated in decreased azithromycin susceptibility in the gonococcus. Isolates were Pro/IB-3 and contained the 17c Lip subtype. Molecular characterization of the mtrR gene revealed a 153 base pair insertion sequence located between the mtrR/mtrC promoter and the mtrC gene. Some isolates also contained a frame shift within the mtrR gene. Transformation of these mutations into an azithromycin-sensitive recipient strain resulted in transformants with MICs as high as 2.0 mg/l and inactivation of the mtrD gene reduced azithromycin MICs 270-fold. These results demonstrated that the mtr mutations were responsible for the increased MICs in these isolates.