The Pseudomonas aeruginosa aacC3 gene was expressed in Escherichia coli after cloning of the single gene behind the strong tac promoter. In the original Pseudomonas strain, aacC3 is preceded by cysC; together they form a single transcription unit. The ribosome-binding site and start codon of aacC3 are involved in a putative intercistronic hairpin, the stability of which interfered with the aminoglycoside resistance level. In Northern blots, full-length transcripts comprising both cysC and aacC3 could not be detected either in the original Pseudomonas strain or in E. coli harboring a plasmid with the cloned operon. In contrast, cysC transcripts were abundant. Cloning of the operon between the tac promoter and a transcription termination signal resulted in higher mRNA levels and phenotypic expression in E. coli. The absence of a transcription termination signal in the wild-type cysC-aacC3 sequence is associated with transcripts of heterogeneous size that were undetected in Northern blots. Our results shed more light on the expression of this gentamicin resistance determinant, although the discrepancies between its expression in E. coli and Pseudomonas are not fully solved.