The presence of Ca2+ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca2+ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl2 in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca2+. This indicates that Ca2+ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca2+, discounting fatty acid mediation of the effects of Ca2+. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca2+.