The effects of EDTA on rat uterine horns were studied and found to consist of contraction followed by relaxation, and inhibition of subsequent contraction in response to acetylcholine. An hypothesis was formulated to explain these actions. The contraction is attributed to complexing and consequent decrease in the activity of magnesium in or near the membrane, accompanied by unstabilization of the membrane following removal of calcium from sites at which it exerts a stabilizing action. The relaxation is attributed to similar complexing of membrane calcium bound to other sites from which it is released during excitation to cause contraction. Na4EDTA was much more effective in evoking contractions than Na2EDTA; this is attributed to the competition between hydrogen and magnesium.Experiments in which ions in the bathing medium were varied supported this general hypothesis. Furthermore, MgEDTA was found to produce only relaxation, while CaEDTA was found to produce little or no effect; mixtures had intermediate effects. The calcium-depleted membrane was found to compete effectively with CaEDTA for its calcium. Similar studies were carried out with DTPA (diethylenetriaminepentaacetic acid) and similar results obtained. DTPA was more dependent than EDTA on a high pH, as expected from the greater affinity of this material for hydrogen ions. EGTA (ethylene bisglycol (β-aminoethylether) tetraacetic acid), a substance with equivalent ability to complex calcium and less ability to complex magnesium relative to EDTA or DTPA, was ineffective in evoking contraction unless magnesium was omitted from the medium; however, it did inhibit responses to subsequent acetylcholine. None of these complexing agents produced significant contractions when strontium was substituted for calcium.Other complexing agents unrelated to EDTA were studied as well. Desferrioxamine (DFO) caused contractions when added at a pH of 9 or more. It had little inhibitory action on acetylcholine-evoked contractions. Oxalate was relatively ineffective in evoking contraction or inhibition of responses to acetylcholine. Both these sets of findings are compatible with the hypotheses for EDTA when considered in the light of the complexometric characteristics of DFO and oxalate. 1,10-Phenanthroline, which complexes neither magnesium nor calcium effectively, evoked no contractions, but did inhibit contractions in response to other agents by a mechanism apparently unrelated to calcium.