We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coli were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.