Temperature sensitive (ts) mutants of methanol-utilizing Hansenula polymorpha NTU-AM-P5 were isolated by UV irradiation, EMS and/or NTG treatment. They grow normally at permissive temperature (PT) 38 degrees C, but can not grow at restrictive temperature (RT) 46 degrees C. From chemical composition analysis, it was found that ts mutants NTU-AM-L2 and NTU-AM-L3 had higher RNA content than the others; while ts mutant NTU-AM-E19 had the highest protein content among the isolated strains. Leucinyl-tRNA synthetases activity was the highest among the twenty aminoacyl-tRNA synthetases in both wild type and their ts mutants. When the cells were shifted from PT to RT for 12 h, total aminoacyl-tRNA synthetase activity decreased in all tested strains. Leucinyl-tRNA synthetase of ts mutant NTU-AM-E10 decreased 91.23%. At RT, it was found that ts mutants NTU-AM-E10 and NTU-AM-E20 were defective in DNA synthesis; ts mutants NTU-AM-E15, NTU-AM-E20, NTU-AM-N37 and NTU-AM-m5 were defective in RNA synthesis; ts mutants NTU-AM-E10, NTU-AM-E20 and NTU-AM-m5 were somewhat defective in protein synthesis; while ts mutants NTU-AM-L2 and NTU-AM-L3 did not belong to any one of the above classifications.