The antitumor antibiotic sparsomycin, produced by Streptomyces sparsogenes, is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive Streptomyces lividans with an S. sparsogenes library. Resistance was linked to the presence of a plasmid containing an S. sparsogenes 5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to srd, an open reading frame of 1,800 nucleotides. Ribosomes from S. sparsogenes and the S. lividans-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier.