The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by the $quot;in-situ colony hybridization technique$quot; using synthetic oligonucleotide as a probe. Pstl digested chromosomal DNA was ligated into the Pstl site of pBR322. The ligation mixture was used to transform E. coli and the transformants were screened by the in-situ colony hybridization technique. Out of 3,500 colonies screened, one colony was hyridized to the probe. The recombinant DNA of this positive clone was named pS113. Digestion of pS113 by restriction endonucleases showed that the size of the inserted DNA was about 4.6 Kb and the restriction map was nearly like that of Jacobs`. To express the gene in Bacillus, Pstl digested pS113 was ligated into the Pstl site of pMK4. The ligation mixture was used to transform B. licheniformis and the transformants were assayed on casein plate for protease secretion. We obtained a colony having a distinctively large halo.