Nitric oxide (NO) has various physiological functions. However, uncontrolled overproduction of NO can be toxic in many pathologic conditions involving inflammatory tissue damage. In the present study, we examined effects of 23,24-dihydrocucurbitacin D (DHCD) isolated from the root of Bryonia alba L. on macrophage NO generation. DHCD (<80 microM) effectively abolished NO generation from macrophages activated with lipopolysaccharide and interferon-gamma. DHCD decreased the levels of protein and mRNA for inducible NO synthase (iNOS). DHCD potently blocked nuclear factor-kappaB (NF-kappaB) activation, a process necessary for transcriptional activation of iNOS. These results suggested that DHCD inhibited NO generation by blocking NF-kappaB activation and iNOS gene transcription. Because NF-kappaB activation is necessary not only for NO generation but also for many inflammatory processes, DHCD and its derivatives could be developed as anti-inflammatory drugs.