The aac(6')-Ib' gene from Pseudomonas fluorescens BM2687, encoding an aminoglycoside 6'-N-acetyltransferase type II which confers resistance to gentamicin but not to amikacin, was characterized. Nucleotide sequence determination indicated total identity between aac(6')-Ib' and the aac(6')-Ib gene from Pseudomonas aeruginosa BM2656 [1] with the exception of a C-to-T transition that results in a serine to leucine substitution at position 83 of the deduced polypeptide. The aac(6')-Ib gene specifies a type I enzyme which confers resistance to amikacin but not to gentamicin [2]. It thus appears that the point mutation detected is responsible for enzymic altered substrate specificity.