A novel gene encoding a 6'-N-aminoglycoside acetyltransferase, aac(6')-In, has been cloned and sequenced from Citrobacter freundii 13996-19, a clinical isolate from Venezuela. This gene mediates resistance to amikacin, 2'-N-ethylnetilmicin, isepamicin, kanamycin, netilmicin, and tobramycin. The aac(6')-In gene is 573 nucleotides in length and encodes a putative protein of 190 amino acids. AAC(6')-In is most closely related to AAC(6')-Im and AAC(6')-Ie, demonstrating 64.4% and 62.3% similarity, respectively, at the protein level, suggesting these proteins share a common ancestor. The aac(6')-In flanking sequences demonstrated homology to integron- and transposon-related elements which are often found associated with resistance determinants. Hybridization studies performed with an intragenic probe specific for aac(6')-In indicate that this gene is prevalent within Venezuela but has not been observed outside of the country. Furthermore, the aac(6)-In gene was found in 10 different species of gram-negative bacteria.