Chemical nuclease, NiCR[(2,12-dimethyl-3,7,11,17-tetraazabicyclo[11,3,1]heptadeca-1,(17),2,11,13,15-pentaene) nickel(II) perchlorate] was prepared and used in the structure determining study of 5S rRNA from Pseudomonas alcaligenes. The reaction condition of the complex and the cleavage sites of 5S rRNA were investigated. Strong cleavage was observed at U40, C88 and G89 spontaneously of denatured and renatured 5S rRNA. A weak cleavage at U63 was also observed. The cleavage at G100 occurred only in denatured 5S rRNA and the strength of cleavage was affected by each step of modification. No cleavage was observed at G100 in renatured 5S rRNA. This means that G100 residue is located in the site that is not accessible to NiCR in the three dimensional structure of renatured state and exposed to NiCR in denatured state.