Gram-negative bacteria are the most important infective agents in hospitalized patients. Aminoglycosides are still important in the treatment of infections caused by Gram-negative bacteria. In recent years a new resistance mechanism emerging by methylation of the bacterial 16S rRNA has been reported. This mechanism, causing high-level aminoglycoside resistance, is coded on plasmids and easily transferred among Gram-negative bacteria. The purpose of this study was to investigate aminoglycoside resistance mechanism acquired by 16S rRNA methylase enzymes in Gram-negative bacteria. With microdilution method high-level aminoglycoside resistance was detected only in one Acinetobacter baumannii strain out of 112 Gram-negative bacteria. Methylases enzymes genes were not observed by polymerase chain reaction+agarose gel electrophoresis in this strain. 16S rRNA methylase genes are usually found with beta-lactamase genes and confer high-level resistance against most of the aminoglycosides. Therefore, investigation of this resistance mechanism by further studies in our country is required.