Systems and methods for performing online extracorporeal photopheresis of mononuclear cells are disclosed. Whole blood is removed from a patient and introduced through a processing set into a separation chamber to separate the desired cell population from the blood. The separated cell population is processed through the set which is associated with a treatment chamber where the cells are treated. Once treated, the cells are returned to the patient. The processing set remains connected to the patient during the entire ECP treatment procedure and provides an online, sterile closed pathway between the separation chamber and the treatment chamber.