We visualized the course of nucleotidyl transfer reaction by human DNA polymerase η (Pol η) using time resolved protein crystallography. The reaction was initiated by exposing Pol η-DNA-dATP crystals to 1 mM Mg<sup>2+</sup> at pH 7.0 and stopped at various time points by freezing. The substrates and two Mg<sup>2+</sup> ions are aligned for reaction within 40 s. Transient electron densities indicate that deprotonation and an accompanying C2-endo to C3-endo conversion of the nucleophile 3-OH are rate limiting. A third Mg<sup>2+</sup> ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.