The intracellular distribution and several properties of hexokinases type I purified to homogeneity from human placenta and rat brain were compared. The specific activity of the human enzyme was 190 +/- 5 U/mg protein; 140 +/- 5 U/mg protein that of the rat hexokinase. Comparative peptide mapping after limited tryptic digestion indicates a similar domain structure, however analogous experiments performed in the presence of substrates or effectors of the enzyme provide evidence of significant differences among hexokinases. Similarly, immunological studies with polyclonal and monoclonal antibodies while confirming some common epitopes also disclose important differences that cannot be expected on the basis of amino acid composition and of an in vivo identical function. These results are consistent with suggestions by several investigators that amino acid substitutions in mammalian hexokinases have occurred at a relatively fast rate during hexokinase type I evolution.