This experiment addressed the hypothesis that 6-phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4°C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P<0.001) from 0.84μmol/g at d 0 to 6.54μmol/g at d 4. Fructose-6-phosphate and glucose-6-phosphate increased (P<0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33μmol/g) and was elevated to 45.9μmol/g by d 4. In the second experiment, conversion of [U-(14)C]glucose to lactate, glycogen, and CO(2) was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U-(14)C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123nmol glucose utilized per 100mg muscle per h; P=0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO(2) in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.