We evaluated the ventilation ability of two types (plug-type and cap-type) of culture-stoppers having standard air permeability. The culture-stoppers were evaluated using the circulation direct monitoring and sampling system with CO(2)concentration in the gas phase of a shake-flask culture as an index. The half-lives of CO(2)in the headspace of the shake flask with the plug-type and cap-type stoppers were about 51.5 min and about 30.3 min, respectively. Based on these half-lives, we formulated a model equation to simulate the behaviour of CO(2)with different culture-stoppers. After validating the model equation by shake-flask culture withSaccharomyces cerevisiae, we investigated the effect of different ventilation abilities of the culture-stoppers on the growth ofPelomonas saccharophilaandEscherichia coli: the sensitivity of the culture-stopper to the ventilation ability was dependent on the microorganism species. In the case ofP. saccharophila, when the plug-type culture-stopper was combined with controlled CO(2)concentration (6%) in the flask, the maximum yield increased by twofold compared to that of the control. This study shows the importance of ventilation in headspace and conventional culture-stoppers during the shake-flask culture of microorganisms. The problems that may occur between the conventional shake-flask culture approach using a breathable culture-stopper and the next-generation shake-flask culture without a conventional culture-stopper were clarified from the evaluation of gas-permeable culture-stoppers. The importance of controlled gaseous phase in the headspace during shake-flask culture of the microorganisms was also elucidated.