A simple and rapid new method is presented to determine the CO 2/O 2 specificity factor for ribulose_1,5_bisphosphate(RuBP) carboxylase/oxygenase (Rubisco). In principle, the quantitative formula, which intrinsically resulted in error or enormous error in all reported methods, was improved to circumvent the problem. In practice, the complicated procedure with radiolabelling and chromatographic separation of 3_phosphoglycerate and 2_phosphoglycolate was substituted by one_step procedure without radiolabelling and chromatographic separation to decrease the random error. The effects of pH, temperature and ionic strength on accuracy of the data and the reliability of the calculation of the CO 2/O 2 specificity factor are also discussed.