The pullulanase gene from Clostridium thermosulfurogenes (DSM 3896) was cloned and expressed in Escherichia coli with pUC18 as cloning vector. Two clones showed expression of amylolytic enzymes which were active at high temperatures. One of the recombinant plasmids (pCT3) containing a 5.3 kbp insert coded for the pullulanase gene; the other (pCT4, 4.4 kbp insert) carried the same alpha-amylase gene as the previously described plasmid pCT2 (2.9 kpb insert, 7). The pullulanase gene was efficiently transcribed in E. coli, apparently using its own promoter; the enzyme was not secreted into the medium. No difference in the temperature optimum and thermostability between the original and the heterologously expressed (in E. coli) enzyme could be found.