Abstract Previous work on the evolution of the phosphofructokinase (PFK) has shown that this key regulatory enzyme of glycolysis has undergone an intricate evolutionary history. Here, we have used a comprehensive data set to address the taxonomic distribution of the different types of PFK (ATP-dependent and PPi-dependent ones) and to estimate the frequency of horizontal gene transfer (HGT) events. Numerous HGT events appear to have occurred. In addition, we focused on the analysis of sites 104 and 124 (usually Gly 104+Gly 124 or Asp 104+Lys 124), known to be involved in catalysis (J. Biol. Chem. 275 (2000) 35677). It revealed the existence of numerous sequences from distantly related species carrying atypical combinations of amino acids. Several adaptive changes of phospho-donors, probably requiring a single mutation at position 104, have likely occurred independently in many lineages. The analysis of this gene suggests the existence of a high rate of both HGT and substitution in its active sites. These rampant HGT events and flexibility in phospho-donor use illustrate the importance of tinkering in molecular evolution.