The L-asparagine operon of Rhizobium etli was cloned and sequenced. Sequence analysis showed four adjacent open reading frames which were designated as ansR, ansP, ansA and ansB. The ansR and ansP genes encoded proteins similar to a transcriptional repressor and an L-asparagine permease, respectively. By Tn5 mutagenesis and complementation analysis we identified the ansA product as a thermolabile asparaginase, and the ansB product as an aspartase. An asparagine-inducible transcript covering ansP, ansA and ansB was detected by reverse transcription (RT)-PCR, indicating that these genes are organized in an operon. Introduction of the R. etli ans operon into Sinorhizobium meliloti induced growth with asparagine as the sole carbon and nitrogen source, suggesting that the ans operon plays the same physiological role in both bacteria. The product of the R. etli ansA gene showed no sequence similarity with previously reported microbial asparaginases, this protein seems to be an atypical asparaginase which evolved apart from bacterial and yeast asparaginases.