It has become increasingly evident that horizontal gene transfer is central to microbial activities that influence our health and the environment. The influence of horizontal gene transfer in antibiotic production has not been well documented, although there is indirect evidence that it occurs. We carried out competitive co-cultures between a multi-antibiotic resistant mutant of a strain of Rhodococcus fascians that does not produce an antibiotic and a strain of Streptomyces padanus that is a highly stable actinomycin producer. A strain (307CO) of the Rhodococcus emerged from one such culture with the concomitant elimination of the Streptomyces. Bioassays of the Rhodococcus showed that it produces one or more antibiotics. Genomic analysis revealed that the Rhodococcus 307CO harbors a large segment of DNA derived from the Streptomyces strain, and there was a correlation between antibiotic production and the presence of the Streptomyces DNA in the Rhodococcus 307CO. We have isolated two antibiotics, named rhodostreptomycin A (1) and B (2), from culture broths of Rhodococcus 307CO. These appear to be two isomers of a new class of aminoglycosides. Rhodostreptomycins A (1) and B (2) both had molecular formulas of C22H40N8O13, determined via high-resolution electrospray ionization time-of-flight mass spectrometry. The two isomers differ in the configuration of the carbon atom bearing the hydroxyl group in the oxazine (C6 in Strp), which is R in rhodostreptomycin A (1) and S in rhodostreptomycin B (2). Structural elucidation was performed using tandem ion trapping mass spectrometry and NMR experiments including proton, carbon, GHMBC, DQCOSY, GHMQC, and ROESY or NOESY. Rhodostreptomycins exhibited good antibiotic activities against an extensive range of Gram-negative and Gram-positive bacteria, including Helicobacter pylori in conventional disk assays run on microbial lawns. The activity of rhodostreptomycin B (2) was more potent than that of rhodostreptomycin A (1). No evidence of activity against eukaryotes such as Saccharomyces cerevisiae or cytotoxicity against human leukemia (HL-60) was observed. In conclusion, new isomeric antibiotics produced by Rhodococcus 307CO, and consequently named rhodostreptomycin A and B, have been isolated and characterized by mass spectrometry and NMR spectroscopy. Surprisingly, the antibiotics biosynthesized in the Rhodococcus following horizontal gene transfer from the Streptomyces are aminoglycoside antibiotics and differ widely in the structure from actinomycins, polypeptide antibiotics that are produced by Streptomyces. Knowledge concerning rhodostreptomycins production from Rhodococcus, especially with regard to the role of gene transfer, could be an attractive research challenge from academic and industrial points of view.