The naphthgeranines A to E were previously isolated from Streptomyces violaceus (strain Tii 3556) in a chemical screening program. In this study, strain Tii 3556 was included in a screening program for new secondary metabolites using HPLC, diode array multiwavelength monitoring and a UV-visible absorbance spectral library. Besides phenazine-1-carboxylic acid, methyl 1-phenazinecarboxylate and the new natural product 1-(3-indolyl)-2,3-dihydroxypropan-1-one (1, which was previously only known as a synthetic substance), a new compound with a UV spectrum similar to naphthgeranine E (2) but a different HPLC retention time was detected and named naphthgeranine F (3). Strain Tii 3556 was cultivated in medium containing 2% mannitol and 2% soybean meal; naphthgeranine F concentration was similar to that of E (<1 mg/liter) after 120 hours of fermentation, with naphthgeranine C as the main compound (9 mg/liter). No C-, N- or P-repression of antibiotic production was observed. Naphthgeranine F was isolated from the culture filtrate via Amberlite XAD-16 column chromatography, ethyl acetate extraction, oxalic acid-treated silica gel chromatography, Sephadex LH-20 chromatography and preparative reversed-phase HPLC, yielding a dark-red amorphous substance. Its structure was elucidated by HREI-MS (molecular ion peak at m/z 368.0896, consistent with the calculated value for C20H16O7) and NMR analyses (1H, 13C, COSY, HETCOR, COLOC), revealing a 5-hydroxy-1,4-naphthoquinone skeleton with structural variations from naphthgeranine E in ring D (lack of a phenolic hydroxy group at C-3 and an additional hydroxy group at C-14 forming a hydroxymethylene group), which was confirmed by a COLOC experiment. Physicochemical properties of naphthgeranine F are shown in Table 1, and 13C and 1H NMR chemical shifts are shown in Table 2. Antimicrobial activity testing showed that naphthgeranine F had weak activity against Gram-positive bacteria (Bacillus subtilis ATCC 6051, Bacillus brevis ATCC 9999, Streptomyces viridochromogenes Tii 57) with MIC values similar to those of naphthgeranine C, but no activity against Gram-negative bacteria, yeasts or other fungi at a concentration of 1 mg/ml. Structural variations within the naphthgeranine family result from high oxygenase activity directed towards the mevalonate-derived part of the molecule. The center of chirality at C-5 of naphthgeranine F requires stereospecifically acting enzymes, leading to the assumption that strain Tii 3556 may be suited for biotransformations of steroids.