Two genes encoding for aminoglycoside-(3)-N-acetyltransferases (AAC(3)s) of different substrate patterns, present in multiresistance plasmids of hospital strains of Serratia marcescens and Escherichia coli isolated from urine, have been cloned and characterized. The first, aacC1 with AAC(3)I activity, contained a 531 base pair open reading frame which encodes a polypeptide of 177 aminoacids and 19,392 daltons, confirmed by minicell analysis. Its sequence differed from previously published work in four positions. Three of the changes did not alter the aminoacid sequence while the fourth was a substitution of an alanine by a proline. The second gene, an AAC(3)II encoded by aacC2, resulted from the translation of an 858 base pair open reading frame, which encoded a 286 aminoacid polypeptide of 31,574 daltons and was identical to those from plasmids isolated in Germany and the United States. However, the homology was broken in a position between the -10 and -35 promoter sequences, which resulted in different -35 hexanucleotides and levels of resistance conferred. The assay of both genes as molecular probes has revealed their specificity with respect to other aac genes, although their usefulness was limited in the case of aacC1 derived sequences to isolated plasmid DNA, since it hybridized under stringent conditions with chromosomal DNA of some strains of E. coli.