We have developed a dual expression system for the simultaneous overexpression of two proteins in Bacillus subtilis. Two candidate genes, xylanase (xynA) and glucanase (bglS) from B. subtilis strain 168, which were engineered with independent Shine-Dalgarno (SD) sequences, were cloned in tandem into a transfer vector, which was then transformed into B. subtilis strain 1A304 (phi105MU331). The genes were under the transcriptional control of a strong promoter of a bacteriophage, 105, where transcription was initiated upon thermal induction. Six constructs were made to compare the factors that affected the yields of the gene products. The expression level of each candidate gene was found to correspond to its position relative to the phage promoter, irrespective of the identity of the insert. The lower expression level of the second insert might have been due to limited resources for protein synthesis, a short half-life of the mRNA, or an early termination of the RNA polymerase. Curiously, gene duplications in tandem did not lead to further increase in production. (C) 2002 Elsevier Science (USA). All rights reserved.