In phosphate buffers below pH 7, rabbit skeletal muscle phosphofructokinase loses activity as a function of time. The inactivation is reversible and becomes more extensive at lower temperature, lower pH values, and lower enzyme concentrations. It is correlated with changes in the molecular weight of the enzyme as measured by light scattering. The effect of temperature between pH 6 and 7 shows the characteristics of enzyme systems described as cold labile. In general the loss of activity is biphasic but the phases do not appear to be directly correlated with individual molecular events. A mechanism consistent with the inactivation data as a function of enzyme concentration involves three reversible steps: dissociation of the active enzyme to an inactive form which can either repolymerize to an inactive form or isomerize to a different inactive form. ATP addition early in the inactivation process results in rapid partial inactivation (and depolymerization). ATP addition late in the inactivation process results in partial reactivation (and repolymerization). The results may be explained in terms of ATP binding with different affinities to the different forms of the enzyme arising in the inactivation process.