The two genes encoding precursors of co-produced endo-1,4-beta-D-xylanases, Xyn6 and XynB, were isolated from Aspergillus niger IBT-90 by using RT-PCR technique and expressed in Pichia pastoris under the control of the alcohol oxidase I promoter. The secretion was driven by the Saccharomyces cerevisiae alpha-mating factor fused to mature xylanases and by the native 27-amino acid leader peptide of Xyn6 or 37-amino acid signal of XynB when the entire open reading frames of proteins were cloned. The secretion level of XynB directed by alpha-factor estimated at 140 mg l(-1) was comparable to 150 mg l(-1) with its own leader peptide; whereas in case of Xyn6, the yield was up to 180 and 220 mg l(-1), respectively. The N-termini of active recombinant Xyn6 and XynB indicated that their endogenous pre(pro)signals are effectively recognised and correctly processed in P. pastoris, like alpha-factor. These findings should contribute to develop the possibilities of application of the alternative secretion signals in P pastoris. The initial studies revealed the different pH optima of Xyn6 (3.5) and XynB (5.0). The further analysis of individual gene products should enable to clarify the role of a particular enzyme in a complex xylanase system of A. niger. (c) 2005 Elsevier Inc. All rights reserved.