Transient absorption measurements of the pheophytin a anion band and a band bleach region using preferential excitation of P680 are performed on isolated photosystem II reaction centers to determine the effective rate constant for charge separation. A novel analysis of the Q(x) band bleach region explicitly takes the changing background into account in order to directly measure the rate of growth of the bleach. Both spectral regions reveal biphasic kinetics, with a ca. (8 ps)(-1) rate constant for the faster component, and a ca. (50 ps)(-1) rate constant for the slower component. We propose that the faster component corresponds to the effective rate constant for charge separation from within the equilibrated reaction center core and provides a lower limit for the intrinsic rate constant for charge separation. The slower component corresponds to charge separation that is limited by slow energy transfer from a long-wavelength accessory chlorophyll a.