Phosphoryl transfer is a key reaction in many aspects of metabolism, gene regulation, and signal transduction. One prominent example is the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which represents an integral part of the bacterial sugar metabolism. The transfer between the enzymes IIA (Glc) and IIB (Glc) in the glucose-specific branch of the PTS is of particular interest due to the unusual combination of donor and acceptor residues involved in phosphoryl transfer: The phosphoryl group is initially attached to the Nepsilon2 atom of His 90 in IIA (Glc) and then transferred to the S gamma atom of Cys 35 in IIB (Glc). To gain insight into the details of the transfer mechanism, we have performed a QM/MM simulation which treats the entire active site quantum-mechanically. The transfer has a high dissociative character, and the Nepsilon2-P bond gets immediately destabilized after complex formation by numerous interactions formed between residues of IIB (Glc) and the phosphoryl group. The final formation of a tight S gamma-P bond is accompanied by a reorientation of the side chain of the phosphoryl donor. This reorientation results in the loss of interaction between the imidazole ring and the phosphate group thus hindering the reverse transfer. A comparison to the transfer in protein tyrosine phosphatases, which also use a cysteine as acceptor of the phosphoryl group, reveals significant similarities in the conformation of the active site, the energy profile of the reaction, and in the pattern of interactions that stabilize the phosphoryl group during the transfer.