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The conditions for maximal activity of ethanolamine phosphokinase from rat liver were determined. All of the activity was located in the supernatant fraction after centrifugation at 100 000 × g for 60 min. The enzyme activity had a pH optimum at 8.5 and an apparent Km for ethanolamine of 1·10−4 M at an ATP and Mg2+ concentration of 3.0 mM. The enzyme uses the Mg-ATP complex as substrate and is inhibited by free ATP. Choline inhibits ethanolamine phosphokinase. Maximal inhibition is obtained at choline concentrations of 0.4 mM. Treatment of the supernatant with Sephadex G-25 or by dialysis causes an increase in the maximal amount of inhibition obtained with choline. The inhibition by choline is non-competitive with ethanolamine and competitive with ATP. Ethanolamine phosphokinase is inhibited by N,N-dimethylethanolamine and N-methylethanolamine but not by betaine, phosphoryl choline, CDP-choline or phosphorylethanolamine. The activity of ethanolamine phosphokinase is low in −5-day fetal liver. The activity increases from −5 days to −2 days and drops at 1 day after birth. An endogenous inhibitor of ethanolamine phosphokinase is present in all preparations but at different levels. The drop in activity in 1-day-old animals is due to the presence of higher amounts of inhibition by the endogenous inhibitor within the preparation. Evidence indicates that the endogenous inhibitor is choline.