CO2 and O-2 in the Sakaguchi flask headspace during culture was monitored via circulation direct monitoring and sampling system (CDMSS), a device with circulation bypass system. In static culture with Saccharomyces cerevisiae (circulation rate, 50 mL/min), a vertical CO2 concentration gradient (maximum gap similar to 2% (v/v) [height from the bottom of flask 45 mm, 7%; 155 mm, 5%]) in the Sakaguchi flask headspace was observed; no concentration O-2 gradient was observed. However, shake flask culture showed vertical gradient concentrations for both CO2 and O-2 (maximum gap of CO2 and O-2 concentrations: 2 and 4% [heights from the bottom of flask 115 mm, 6.0 and 9.5%; 175 mm, 4.0 and 13.5%], respectively). When the CDMSS circulation rate in the Sakaguchi flask headspace was 300 or 400 mL/min, the gaseous environment was uniformly distributed so that no vertical gradient concentration was observed. In shaking culture with Escherichia coli under these conditions, CO2 was accumulated at high concentrations in the headspace and culture broth (maximum values 8%, in the headspace; 120 mg/L, in the culture broth). Most of the accumulated CO2 in the headspace could be removed by inserting a column packed with CO2 adsorbent at the bypass port of the CDMSS gaseous circulation. Thus, dissolved CO2 was maintained at a lower concentration, and the final UOD (unit optical density) value of culture was increased compared with that of the control. This study is the first to demonstrate that vertical gradients of CO2 and O-2 concentrations exist in the headspace of Sakaguchi flask during culture.