In an effort to better understand the factors influencing carbon fixation by Prochlorococcus, and to elucidate the effects of these cyanobacteria on the ocean carbon cycle, the kinetic parameters and isotopic discrimination of form IA ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Prochlorococcus marinus MIT 9313 were determined. The RubisCO genes (cbbL and cbbS) were cloned and expressed in Escherichia coli. Enzyme was purified via ammonium sulfate precipitation, and the optimum pH and temperature, as well as Michaelis-Menton constants, were determined radiometrically. The degree to which this RubisCO discriminates against 13 CO 2 during fixation was determined by the high-precision substrate depletion method. Purified enzyme had a pH optimum of 7.5, was most active between 20uC and 30uC, had a moderate V max (0.41 mmol min 21 mg protein 21 ), and had the highest K CO2 value (0.75 mmol L 21 ) for a form I RubisCO characterized to date. The e value, e 5 1,000[(k 12 /k 13 ) 2 1], for the enzyme was determined to be 24.0% (95% C.I. 5 22.2-25.6%), within the range observed for other form I RubisCOs. This e value is a critical baseline for interpreting the d 13 C values of marine environmental samples, particularly those collected from the open ocean, where P. marinus is responsible for a substantial fraction of carbon fixation.