We investigated the metabolic effects of buffering agents -amino-4-imidazole-propionic acid (Histidine), N,N-bis(2-hydroxyethyl)glycine (bicine), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) on anaerobic energy production (via glycolysis) and conservation of key regulatory enzyme activity, and phosphofructokinase (PFK) throughout prolonged hypothermic hypoxia in porcine hearts. Hearts from 35 to 40 kg pigs were flushed with one of the following five solutions: St. Thomas' Hospital solution (STHS); modified University of Wisconsin (UW) solution; and three solutions containing modified UW plus 90 mM of histidine, bicine, or BES. The hearts were then stored at 4°C for 10 h. After 10 h of hypothermic hypoxia, lactate values were 6.7-12.9 µmol/g higher than control; this reflected an increase in anaerobic end product of 35-67%. The consequences of enhanced anaerobic metabolism were higher ATP, total adenylate, Energy Charge, and ATP/ADP ratios in most of the buffered groups after 4-10 h cold storage; effectiveness of the buffers employed correlated with buffering capacity (BES proved to be the most effective). PFK remained activated throughout most of the 10-h period in hearts stored with buffers and did not undergo the rapid inactivation experienced by hearts stored in STHS. Conservation of PFK integrity with buffering agents was not related to a pH-mediated event; changes in kinetic parameters suggested that this protection was due to an irreversible posttranslational modification, specifically a dephosphorylation event.