Surviving hemiuteri from donor ovariectomized rats were incubated in Krebs-Henseleit buffer, modified in select experiments with respect to Ca2+, Mg2+ and/or K+. These studies demonstrated the following: 1.1. The retrogressed uterus in vitro maintains a substantial concentration gradient, relative to the medium, for both Mg2+ (3.8:1 in 1.2 mM, 38:1 in 0.12 mM Mg2+) and K+ (13:1).2.2. Insulin added in vitro promotes a net increase in the accumulation of Mg2+ and of K+ (7–16%) in uterine muscle. Mg2+ influx into the tissue does not occur by diffusion per se, and it is in general influenced by the same factors as is K+ accumulation. Under the conditions of incubation used, neither the Na+ nor the Ca2+ content was affected by insulin.3.3. Increases in Mg2+, K+ content (insulin effect) are not associated with alterations in the extracellular space (d-[1-3H]sorbitol or Na+ space).4.4. Ouabain (5·10−4 M) added in vitro gives rise to very striking changes in this muscle: (a) Na+ content increases; (b) K+ content decreases, but less than the Na+ increases; (c) Mg2+ content increases to levels as high as that obtained with insulin in the absence of ouabain; (d) no additional effect on Mg2+ accumulation can be shown when insulin is added to a medium that also contains ouabain.5.5. Omission of K+ from the incubation medium mimics the ouabain effect insofar as: (a) Na+ content increases; (b) Mg2+ content increases; (c) no additional effect of insulin added in vitro is obtained.6.6. The interaction of insulin with its receptor in the plasma membrane appears to engage transport systems, including the ion pump [(Na+ + K+)-ATPase (ouabain sensitive; ATP phosphohydrolase, EC 3.6.1.3)]; how increased translocation of cations which influence enzymatic activities, i.e. Mg2+ and K+, may lead to amplification of the insulin signal at the membrane is discussed.