Fruit bodies and mycelia of shiitake mushroom ( Lentinus edodes) have been shown to contain the cholesterol-reducing compound eritadenine, 2( R),3( R)-dihydroxy-4-(9-adenyl)butyric acid. In the search for a production method for eritadenine, shiitake mycelia were investigated in the present study. The mycelia were cultivated both in shake flasks and in bioreactors, to investigate the effects of pH, stirring rate, and reactor type on the production and distribution of eritadenine. Both the biomass and the culture broth were examined for their eritadenine content. In the shake flasks, the final concentration of eritadenine was 1.76 mg/L and eritadenine was equally distributed between the mycelia and the growth media. In the bioreactors, the shiitake mycelia were found to contain eritadenine in relatively low levels, whereas the majority, 90.6-98.9%, was detected in the growth media. Applying a stirring rate of 250 rpm during bioreactor cultivation resulted in the highest eritadenine concentrations: 10.23 mg/L when the pH was uncontrolled and 9.59 mg/L when the pH was controlled at 5.7. Reducing the stirring rate to 50 rpm resulted in a decreased eritadenine concentration, both at pH 5.7 (5.25 mg/L) and when pH was not controlled (5.50 mg/L). The mycelia in the shake flask cultures appeared as macroscopic aggregates, whereas mycelia cultivated in bioreactors grew more as freely dispersed filaments. This study demonstrates for the first time the extra- and intracellular distribution of eritadenine produced by shiitake mycelial culture and the influence of reactor conditions on the mycelial morphology and eritadenine concentrations.