An active cell-free translation system was prepared from Brevibacterium lactofermentum, a Gram-positive bacteria used in molecular cloning and protein expression. The system contained high speed postribosomal supernatant (S 370), purified ribosomes and a tRNA mixture from Escherichia coli, and synthesized polyuridylic acid-directed polyphenylalanine once optimized for mono and divalent ions, time, and temperature. The translation system was evaluated for sensitivity to several translational inhibitors including several N-glycosidase ribosome-inactivating proteins (RIPs) isolated from plants. The pattern of inhibition by RIPs resembled that observed recently for Gram-negative bacteria such as Escherichia coli and Agrobacterium tumefaciens [Girbés et al., J. Bacteriol., 175, 6721-6724 (1993)]. A typical inhibitory type 1 RIP such as crotin 2 promoted depurination of the rRNA, which upon treatment with acid aniline released a fragment of approximately 230 nucleotides. On these grounds, we propose that bacterial ribosome sensitivity to plant RIPs depends on the bacterial ribosome-specific presence of protein recognition domains in the RIP present only in some RIP but not in others.