Contrary to what has usually been assumed, (Na+ + K+)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na+ + Mg2+ to ADP-NH2 and Pi. The activity is ouabain-sensitive and is not detected in the absence of either Mg2+ or Na2+. The specific activity of the Na+ + Mg2+ dependent AdoPP[NH]P hydrolysis at 37 degrees C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K+. The activity is not stimulated by K+, nor can K+ replace Na+ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na+ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5-9 mM. The Km value for AdoPP[NH]P is 17 microM. At 0 degrees C and 21 degrees C the specific activity is 2 and 14%, respectively, of that at 37 degrees C. AMP, ADP and AdoPP[CH2]P are not detectably hydrolysed by (Na+ + K+)-ATPase in the presence of Na+ + Mg2+. In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37 degrees C of 0.0006, 0.006 and 0.07 h-1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg2+ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h-1 at 37 degrees C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na+ + Mg2+ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0 degrees C. On the other hand, determination of affinity in the (Na+ + K+)-ATPase reaction by competition with ATP at low protein concentrations (microgram/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37 degrees C.