Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate.