Streptornyces lividans 66 was isolated from a clone obtained by shotgun cloning through functional complementation of a xylanase-and cellulase-negative mutant using the multicopy vector plJ702. This enzyme, designated xylanase C, has a relative molecular mass of 22 000 and acts on xylan similarly to xylanase B as an endo-type xylanase producing short-chain oligoxylosides. Its specific activity determined at 1100 I U . m g -1 of protein corresponds on a molecular basis to that of xylanase B and is about three times that of xylanase A. The enzyme shows optimal activity at pH 6.0 and 57 ° C, values that correspond closely to those observed previously for xylanase A and B. Xylanase C appears not to be glycosylated and has a p I > 10.25. Its Km and Vmax on birchwood xylan are 4.1 mg.m1-1 and 3.0 lxmol, m i n -1. m g -1 of enzyme respectively. Whereas specific antibodies raised against xylanase A show no cross-reaction with either xylanase B or with xylanase C, the anti-(xylanase C) antibodies react slightly with xylanase B but not with xylanase A. A comparison of hydrolysis products obtained by reacting individually the three enzymes with birchwood xylan showed characteristic endo-activity patterns for xylanases B and C, whereas xylanase A hydrolysed the substrate preferentially into xylobiose and xylotriose. Sequential xylanase action on the same substrates showed synergistic hydrolysis only when endo-xylanase activity was followed by that of xylanase A.