Several different fructan and sucrose hydrolysing enzyme activities were induced in roots and stubble (mainly leaf sheaths) of Lolium perenne L. plants after defoliation. Among those activities, a fructan-β-fructosidase (EC 3.2.1.80) that hydrolyses predominantly β-(2-6)-fructosyl-fructose linkages (6-FEH) was purified from the stubble. The use of the substrate 6,6-kestotetraose and high-performance anion-exchange chromatography with pulsed amperometric detection allowed linkage-specific screening and sensitive analysis of enzyme activity. A 6-FEH was extensively purified to yield one protein band as revealed by one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The 6-FEH was separated from the contaminating β-(2-1)-linkage-specific fructan-β-fructosidase and invertase activities (EC 3.1.2.26) by ammonium sulphate precipitation, lectin-affinity, anion-exchange and size-exclusion chromatography. The purified 6-FEH was a glycoprotein with an apparent molecular mass of 65000, as determined by size-exclusion chromatography, and of 69000 by SDS–PAGE. The 6-FEH had an activity optimum in the range of pH 5·1 to 5·6. Temperatures above 30°C affected the stability of the enzyme activity; however, its temperature stability was increased in the presence of 6,6-kestotetraose. The purified 6-FEH activity hydrolysed the β-(2-6)-linkages in 6,6-kestotetraose and (1&6)-kestotetraose at rates five times faster than the β-(2-1)-linkages in 1,1-kestotetraose and (1&6)-kestotetraose. Fructose up to 50 mM did not affect 6-FEH activity; conversely, sucrose substantially inhibited the enzyme activity. Other disaccharides did not affect 6-FEH. It is suggested that sucrose might modulate 6-FEH activity in vivo.