Charge separation is an essential step in the conversion of solar energy into chemical energy in photosynthesis. To investigate this process, we performed transient absorption experiments at 77 K with various excitation conditions on the isolated Photosystem II reaction center preparations from spinach. The results have been analyzed by global and target analysis and demonstrate that at least two different excited states, (Chl(D1)Phe(D1))* and (P(D1)P(D2)Chl(D1))*, give rise to two different pathways for ultrafast charge separation. We propose that the disorder produced by slow protein motions causes energetic differentiation among reaction center complexes, leading to different charge separation pathways. Because of the low temperature, two excitation energy trap states are also present, generating charge-separated states on long time scales. We conclude that these slow trap states are the same as the excited states that lead to ultrafast charge separation, indicating that at 77 K charge separation can be either activation-less and fast or activated and slow.