A second xylanase-encoding gene (xln) of Streptomyces lividans 66 was cloned by functional complementation of a xylanase/endocellulase-negative double mutant of S. lividans, using the multicopy plasmid pIJ702. Three clones contained a common 3.1-kb DNA fragment encoding the biosynthesis of a 31-kDa xylanase and a 22-kDa protein which was immunorelated. The xylanase represented at least 80% of the total secreted proteins. These three clones also secreted a small amount of the previously reported 43-kDa xylanase which was detected only by using specific antibodies. Most likely, the DNA fragment contained the complete structural xln gene and some regulatory sequences, since the enzymatic activity was repressed by glucose. In the in vitro transcription-translation system, the plasmid pIAF42 encoded an immunoprecipitable 35-kDa xylanase indicating the presence of a 4-kDa signal peptide.