Ceruloplasmin is a multicopper oxidase that contains three type 1 Cu sites and a type 2/type 3 trinuclear Cu cluster. All other multicopper oxidases contain only one type 1 Cu and a trinuclear cluster. In oxidized human ceruloplasmin, one type 1 Cu site is reduced and cannot be oxidized, at least in part due at to a high reduction potential, which is not catalytically relevant. Here we have examined the thermodynamics and kinetics of electron transfer among the five redox-active Cu sites to obtain insight into the molecular mechanism of intramolecular electron transfer and the function of the additional, redox-active T1 Cu site. The redox potentials of the Cu sites of human ceruloplasmin were determined by reductive and poised potential titrations. In pH 7.0 phosphate buffer, the potentials of the type 1, type 2. and type 3 Cu sites are 448, 491, and 415 mV, respectively. Cl- binds to the trinuclear cluster and significantly increases the potentials of the type 2 and type 3 Cu, indicating that Cl- is a physiologically relevant effector of the redox potentials of the Cu sites. Reductive titrations monitored by EPR indicate that the redox potentials of the two redox-active type 1 Cu sites are the same. Upon reduction and reoxidation with O-2, the trinuclear cluster and 50% of the redox-active type 1 Cu are reoxidized. From EPR, this additional electron is distributed equally between the two redox-active type 1 Cu sites. Rapid freeze-quench EPR demonstrated that the rate of electron transfer between the two T1 Cu sites is >150 s(-1), which is faster than the rate of decay of the native intermediate state and indicates that both sites may be catalytically relevant. After reoxidation, the additional electron does not transfer to the trinuclear cluster. Addition of 1-2 equiv of Fe(II) induces ET to the type 2 and T3 Cu sites, indicating that the trinuclear cluster requires at least two electrons to be reduced. Kinetics of reduction of the oxidized enzyme were also studied. Reduction of the type 1 and type 3 Cu sites is fast, while reduction of the type 2 Cu site is slow, indicating that the type 3 Cu pair is reduced by a reduced type I Cu and another Fe(II). These results provide new insight into the molecular mechanism of intramolecular electron transfer in ceruloplasmin. Possible electron-transfer pathways among the Cu sites were examined, and the role of two functional type 1 Cu sites is discussed.