The biotransformation of dehydroepiandrosterone (DHEA) by Gibberella intermedia was investigated. The formation of the main products was monitored by high-performance liquid chromatography (HPLC). Products were purified and identified as 3 beta,7 alpha-dihydroxy-5-androsten-17-one (7 alpha-OH-DHEA) and 3 beta,7 alpha,15 alpha-trihydroxy-5-androsten-17-one (7 alpha,15 alpha-diOH-DHEA) using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analyses. 7 alpha,15 alpha-DiOH-DHEA is a key intermediate for the synthesis of pharmacologically active steroids. A biotransformation process was optimized to obtain a high concentration of 7 alpha,15 alpha-diOH-DHEA. The results showed the optimal biotransformation process under the following conditions: a culture medium containing 15 g/L glucose, 16 g/L yeast extract, 12 g/L corn steep liquor, and 0.15 g/L ferrous sulfate; an initial pH of 6.5; culture temperature of 30 A degrees C; inoculum size of 4 %; 40 mL volume of the culture medium in 250 mL flasks; the addition of 1 % Tween 80 as co-solvent; and the transformation period of 78 h. After optimization, the molar yield of 7 alpha,15 alpha-diOH-DHEA from 5 g/L substrate DHEA was 77.4 %, which was increased by 115 % than the yield obtained in the original bioconversion process.