AbstractAs one of the regulatory gene products in the HIV‐1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from theStreptomycesstrain A92‐308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay‐guided fractionation with gel filtration, normal‐phase and reversed‐phase chromatography yielded six RTA‐active metabolites belonging to the leptomycin family, the known leptomycin A (1), leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L‐156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT‐IR, FAB‐MS,1H‐NMR,13C‐NMR(JMOD), DQ‐COSY, ROESY, HSQC, and HMBC). The configuration ofallCC bonds of1–6was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated leptomycins1–6inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–cand4a–c) of leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on