The antibiotic litmomycin was isolated from a new Streptomyces species, S. litmogenes, with a yield of over 2 g/liter. It is highly active against gram-positive bacteria (MIC 0.25-1.75 μg/ml) but has little or no activity against gram-negative bacteria, mycobacterium, molds or yeast. Litmomycin was produced by fermentation of S. litmogenes on a peanut meal-starch medium for 48 hours, followed by adsorption on charcoal at pH 4.0, filtration, washing with acidic water, air-drying, defatting with n-hexane, acetone extraction, evaporation to dryness, dissolution in dilute ammonia (pH 8-9), acidification, ethyl acetate extraction, and repeated recrystallization from benzene to obtain pure litmomycin (m.p. 223-225°C). The antibiotic has the molecular formula C22H20O10 (M.W. 444, calculated C 59.46%, H 4.54%; found C 59.41%, H 4.72%). Its electron-impact mass spectrum shows fragment ions at m/e 400 (C20H16O9) and 384 (C20H16O8), and the NH3 chemical ionization mass spectrum shows a peak at 447. Other spectroscopic data include UV(EtOH) nm (log ε): 223 (5.30), 286 (4.57), 532 (4.62) and 576 (4.53); IR (KBr) cm⁻¹: 1770, 1600 and 1565; NMR (220 MHz, TFA) δ ppm (J, Hz): 1.25 (d, 7), 1.69 (d, 7), 1.89 (bd, 15), 3.00 (d, 15), ca. 3.02 (m), 3.34 (dd, 18.5), 4.23 (q, 7), 4.51 (d, 8), 5.01 (br. s), 5.34 (q, 7), 5.62 (m). Bioautography comparison indicated litmomycin's behavior closely resembled that of granaticin (from Streptomyces olivaceus). Spectroscopic data matched those of granaticin A, and direct comparison with a granaticin A sample confirmed their identity. Granaticin (litmomycin) has significant antitumor activity against P-388 lymphocytic leukemia in mice (T/C 166% at 1.5 mg/kg) and cytotoxicity against KB cells (ED50 1.6 μg/ml).