The quality control (QC) failure rate in the serum total carbon dioxide (TCO2) test increases at a higher rate than in other tests over time after calibration. The causes of the increased QC failure rate in the TCO2 test were examined. Using a TBA200RF analyzer (Toshiba Medical Systems), the TCO2 of the QC material was measured at 2-hr intervals and was found to decrease by up to 16.5% at 10 hr after calibration. In contrast, using the P-module and D-module analyzers (Roche Diagnostics), the TCO2 of the QC material did not change significantly during 10 hr after calibration. When the TCO2 level of the QC material was measured hourly over 5 hr with the TBA200FR analyzer while the reagent bottle was rotated at 0, 80, 120, 160, or 200 rpm, the rate of decline of TCO2, increased over time after calibration and with increasing reagent cassette rotation. Therefore, in a clinical laboratory using an automated analyzer with a rotating reagent cassette, it is necessary to set a limit to the calibration time interval in order to satisfy the QC goal.