AbstractHot springs located in a geothermal area of central Italy were investigated with respect to the total number of thermophilic microorganisms as well as to the presence of amylolytic and pullulolytic bacteria. A number of motile thermophilic spore‐forming bacteria were isolated from different sites. The aerobic organisms grew optimally at 60 −65°C on yeast extract‐trypticase and/or xylose‐starch supplemented mineral medium and spores were mostly observed on agar plates after prolonged incubation. According to the results based on phenotypic characterization, G + C% and lipid analyses all the aerobic isolates belong to the genusBacillusand are characterized by an unusual G + C content ranging between 33.4 ‐ 38.8. A limited number of anaerobic organisms were also isolated and investigated for the presence of amylolytic and pullulolytic activity. According to physiological tests these isolates were identified asClostridium thermohydrosulfuricum; when grown on starch, they produced thermostable α‐amylases, pullulanases and