Pyrophosphate:fructose-6-phosphate I-phosphotransferase (PFP: EC 126.96.36.199) was purified 260-fold from leaves of etiolated barley seedlings. The purified enzyme consisted of two subunits, with apparent molecular masses of 65 (α) and 60 (β) kDa. Polyclonal antibodies were raised against the denatured PFP protein eluted from an SDS-polyacrylamide gel. The antibodies recognized both denatured and native PFP. Western blots of crude extracts showed that the activity of PFP in barley leaves is correlated to the amount of PFP protein, and that both the α- and the β-subunits are present in near stoichiometric amounts in all investigated tissues. The apparent molecular mass of the boloenzyme. as determined by gel filtration chromatography, was dependent on the presence of pyrophosphate. In absence of pyrophosphate. barley PFP elutes as a heterotetramer whereas it elutes as a heterooctamer in the presence of 20 mM pyrophosphate. Pure PFP obtained by gel filtration chromatography in the presence of 20 mM pyropnosphaie reached a specific activity of 28 U mg−1. Barley PFP was characterized with respect 10 kinetic properties in the forward direction (use of PP1) and in the reverse direction (formation of PP1). The affinity for the activator Fru-2.6-P2: was very high, with an estimated K3 of 2.8 nM when PFP activity was assayed in the forward direction.