Aims: To isolate and identify dextran-degrading organisms from sugar mill and compost samples, and to examine the diversity of the dextranolytic enzymes produced.Methods and Results: Fifteen dextranolytic prokaryotes were purified at various temperatures from sugar-mill or compost samples, using indicator plates containing blue dextran. A 16S rRNA gene sequence analysis showed that 12 isolates purified at 40, 50 or 70degreesC were closely aligned to Paenibacillus spp. The three isolates purified at 60degreesC had identical 16S rDNA sequences, with highest affinity to Bacillus spp. Liquid culture of the 11 isolates purified at 40 or 50degreesC produced dextranolytic activity in the spent media with maximal activity at 40 or 45degreesC under the assay conditions used. Hydrolysis of blue dextran in activity gels showed that the 12 Paenibacillus isolates produced from one to five dextranolytic proteins, ranging from 70 to 120 kDa. Based on 16S rDNA sequence, growth habit in liquid culture and dextranolytic enzyme pattern, the 12 Paenibacillus-like isolates could be differentiated into six distinct groups, one of which was capable of growth at 70degreesC.Conclusions: The Bacillales, especially the Paenibacillus, are a valuable environmental repository for dextranolytic enzymes of diverse size and potentially diverse activity.Significance and Impact of the Study: Dextranolytic enzymes produced by Paenibacillus spp. are an exploitable resource for those interested in modifying the structure of dextrans.