This study demonstrates the effects of oxygen limitations on growth of Atropa belladonna hairy roots in 250-mL shake flasks. Exponential growth was observed only for the first 6-8 d culture; after this time, growth continued with declining specific rate. Increasing the shaking speed and decreasing the medium volume improved biomass production and, to some extent, the specific growth rate of the roots. Direct enhancement of oxygen delivery, either by periodic exposure of the roots to air or oxygen enrichment of the atmosphere, also increased growth rates and/or final biomass levels. These results show that oxygen limitations are likely to affect biomass production and kinetic measurements in shake flask cultures of hairy roots.