In this study, the effect of three carbon sources (mannitol, minced potato and sucrose), two immobilization substrates (alginate and wood cylinders), and three surfactants (Tween 20, Tween 40 and Tween 80), were evaluated on eritadenine production using shiitake (Lentinula edodes) mycelium under submerged cultivation, in shake flasks within 20 days. Eritadenine and biomass were measured by HPLC and gravimetrically, respectively. Alginate immobilization of mycelium promoted significant enhancement of eritadenine yields of 88 mg/L, compared to the control (8.7 mg/L) and wood immobilization (14.8 mg/L). Likewise, eritadenine yields (72.4 mg/L) were enhanced by adding surfactant tween 20 to the broths in 0.5%, than control (8.7 mg/L) without surfactant. Tween 40 and 80 did not improve eritadenine yields, but both produced better biomass values (superior to 5 g/L) than the control (3.9 g/L). All carbon sources (sucrose, mannitol, mince potato, and glucose as control) produced similar low eritadenine yields, with best results (10.2 mg/L) by sucrose, although glucose produced the best biomass yields of 3.9 g/L. Also, carbon sources and the best biomass values did not show significant effect on eritadenine production. pH values in the best eritadenine yielding fermentations went down from 6 to 3-4, but pH had a low correlation with eritadenine yields. Finally, all data obtained in the present study are useful for optimizing culture conditions, towards industrialization of this important health improver metabolite (eritadenine). Key words: Eritadenine, shiitake mushroom, submerged culture, immobilization, surfactants.