The mechanism of resistance to gentamicin and tobramycin in a clinical isolate of Acinetobacter baumannii, in which aminoglycoside-modifying enzymes were not detected, was investigated. For increase of the resistance gene product, DNA prepared the A. baumannii isolate was cloned into pUC18 and introduced into Escherichia coli by transformation. Gentamicin-resistant transformants were screened for aminoglycoside-modifying enzymes. This approach identified two genes encoding AAC(3) and AAD(2") activity, respectively. To determine whether both genes are expressed in the host Acinetobacter strain, we extracted total cellular RNA from this strain, and Northern blots were hybridized with the cloned AAC(3) and AAD(2") structural genes. mRNA transcribed from the AAC(3) gene alone was detected. This shows that cloning a functional resistance gene is not sufficient in itself to investigate mechanisms of resistance in bacterial strains without detectable aminoglycoside-modifying activity. Furthermore, this study suggests a potential limitation of antibiotic resistance gene probes for studying mechanisms of resistance.