A method is described for the mild isolation from wheat flour of water-insoluble cell wall material (WIS), containing low amounts of starch and intracellular protein. The isolation procedure was based on the formation of a dough structure from which WIS was released by washing, followed by a wet sieving procedure. A mass balance of the non-starch polysaccharides (NSP) over the various fractions obtained was made, WIS accounted for c. 50% of the total wheat flour NSP. Cold water-soluble cell wall material made up c. 30% of the total NSP. The remaining 20% was found in the starch and gluten fractions and as hot water-soluble cell wall material. WIS was obtained in two fractions, WISI and WIS2, which differed in size. WISI was recovered on a 32 μm sieve, whereas WIS2 passed through it. Composition studies revealed great similarities between WISI and WIS2. The WIS1 fraction consisted of approximately 93% NSP, 2% starch and 2% protein, whereas the WIS2 fraction contained more starch and protein. The protein present in the WIS fractions contained significantly more glycine than the protein in the other fractions. Besides NSP, protein and starch, the WIS fractions contained small amounts of ferulic acid and lipids. Only traces of uronic acids were detected.