To detect mutagenic activity of isepamicin (HAPA-B), a new aminoglycoside antibiotic, we carried out several mutagenicity tests using microorganisms and cultured mammalian cells. Effects of the antibiotic on DNA-lesion induction and repair were examined using the Rec-assay with Bacillus subtilis and a sister-chromatid exchange test with cultured Chinese hamster cells. The drug caused no increase of mutagenicity indices in both tests, suggesting that the antibiotic has no DNA-damaging effect. To further investigate the effect of HAPA-B on gene-mutation, we performed the Ames Salmonella/microsome plate assay and the thioguanine resistance mutation test using cultured Chinese hamster cells. In both tests, the antibiotic induced no increase of mutation frequencies over spontaneous levels. Clastogenic activities of HAPA-B and its effect on chromosome disjunction were examined by the chromosomal aberration test using cultured Chinese hamster cells. After a short- or long-time exposure, the antibiotic induced neither structural chromosome aberrations nor an increase in the number of tetraploid cells. The negative results from these five test systems with different mechanisms strongly suggest the safety of HAPA-B regarding mutagenicity.