Four E. coli-Bifidobacterium shuttle vectors were constructed using Bifidobacterium plasmids, pB44 and pB80. The vectors carry two bifidobacterial promoters, a signal peptide-encoding sequence, sec2, of Bifidobacterium breve, and a transcriptional terminator from hup gene of Bifidobacterium longum. Functionality of the constructs were tested using human FGF-2 gene. The expression of FGF-2 was detected by Western blotting in B. breve transformed with three of the vectors. The highest amount of FGF-2 was produced upon transformation with pESH86, which is a pB80-based plasmid carrying FGF-2 under control of a hup promoter (Phup). Similarly, the level of FGF-2 mRNA transcribed from pESH86 was approximately threefold higher, 882 +/- 70 AU (arbitrary units), when compared to those transcribed from pB44-based pESH46 (Phup) (289 +/- 65 AU) and pESH47 (Pgap) (282 +/- 37 AU). These results suggest the vectors have the potential for production of exported fusion proteins in bifidobacteria and the expression levels can be regulated through the employment of different bifidobacterial promoters and/or replicons.