We investigated antibacterial activities of combination uses of isepamicin (ISP) and beta-lactams in vitro against Klebsiella pneumoniae and Enterobacter cloacae, and the following conclusions were obtained. 1. ISP+cefazolin, ISP+cefotiam and ISP+flomoxef against K. pneumoniae and ISP+piperacillin, ISP+ceftazidime, ISP+aztreonam, ISP+imipenem and ISP+panipenem against E. cloacae showed strong combined effects. 2. The minimum inhibitory concentrations (MICs) of these combinations were low due to the dependence of ISP concentrations. Strong antibacterial activities were observed at sub-MIC levels of ISP. These combined effects were stronger than those against Staphylococcus aureus described in the first report at sub-MIC levels of ISP. 1/4 MIC approximately 1/8 MIC of ISP showed enhanced activities of beta-lactams. Similarly strong combined effects were observed against both beta-lactam-sensitive and -resistant strains.