Recent ³¹P-NMR saturation transfer measurements of flux between P_i and ATP in the perfused rat heart (Kingsley-Hickman, P., Sako, E.Y., Andreone, P.A., St. Cyr, J.A., Michurski, S., Foker, J.E., From, A.H.L., Petein, M. and Ugurbil, K. (1986) FEBS Lett. 198, 159–163) have given a P/O ratio (mols ATP synthesised/atoms oxygen consumed) which was close to 6. This anomalously high value was attributed to exchange in the reaction catalysed by the mitochondrial F₁F₀-ATP synthase. We show here that this exchange could also be catalysed by the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. ³¹P-NMR saturation transfer measurements of the exchange catalysed by these enzymes in vitro, under conditions designed to mimic those present in the perfused rat heart, have shown that they could catalyse a quantitatively significant P_i-ATP exchange in vivo. A three-site exchange model is used to investigate the effects of P_i-ATP exchange on saturation transfer measurements of the reverse flux in the creatine kinase reaction. A discrepancy in the measured and forward and reverse fluxes in this reaction has been attributed previously to the participation of the γ-phosphate of ATP in other exchange reactions.