Several kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase from different species were measured and compared. The CO2/O2 specificity (VcKo/VoKc) was found to be about 80 in the enzymes from several C3 species and two C4 species. Specificity values of 58 and 70, respectively, were found in enzymes from the C4 plants Setaria italica and Sorghum bicolor. Two enzymes from cyanobacteria had values of about 50. Substitution of Mn2+ for Mg2+ reduced the CO2/O2 specificity by a factor of about 20 for all enzymes except that of Rhodospirillum rubrum, which was reduced by a factor of 10. Values for KMg2+(apparent) measured at 102 microM CO2 were found to vary by a factor of 8 between different RuBP carboxylase/oxygenase enzymes. Enzymes with high KMg2+(apparent) values generally had high Michaelis constants for CO2. The rate of CO2/Mg2+ activation was inhibited by RuBP in all enzymes, although the concentration of RuBP required to inhibit activation in the enzyme from the cyanobacterium Aphanizomenon flos-aquae was increased by an order of magnitude compared to other higher plant structural-type enzymes. The wide variation found in the kinetic properties of RuBP carboxylase/oxygenase isolated from diverse species appears to be determined in part by past evolutionary pressures and the present physicochemical environment in which the enzyme functions.