The endoribonuclease III (RNase III), encoded by the rnc gene, is an important enzyme for RNA metabolism. In this report a chromosomal fragment containing the rnc gene from Lactococcus lactis was cloned and its expression was analyzed. Complementation assays performed in Escherichia coli demonstrate that the lactococcal RNase III (Lac-RNase III) is able to process rRNAs and to regulate the levels of polynucleotide phosphorylase (PNPase). These results demonstrate that the lactococcal enzyme is able to substitute the Ec-RNase III not only in the rRNA processing, but also in the processing of mRNAs. The amount of lactococcal rnc transcript in an E. coli Δrnc strain was 3.3-fold higher than in the wild type strain, suggesting that the E. coli RNase III triggers the degradation of the heterologous rnc mRNA. Lac-RNase III is able to cleave an in vitro synthesized mRNA substrate specific for the Bacillus subtilis homolog. Using this substrate, we standardized an enzymatic assay which allows the specific detection of the endonucleolytic activity of Lac-RNase III in L. lactis and E. coli crude extracts.