The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.