One hundred Peptostreptococcus isolates from five species were assessed for their ability to hydrolyze gelatin. Most Peptostreptococcus magnus (95.8%) and Peptostreptococcus micros isolates (79.0%) hydrolyzed gelatin in contrast to Peptostreptococcus asaccharolyticus (8.0%), Peptostreptococcus anaerobius (10.0%), and Peptostreptococcus prevotii isolates (16.7%). Gelatin hydrolysis in Peptostreptococcus magnus and Peptostreptococcus micros isolates correlated (r = 0.80; P = 0.0019) with more aminopeptidases produced than Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, or Peptostreptococcus prevotii. The five species were further classified into three groups using the extended Tukey test (P < 0.0001) based on the mean percentage of aminopeptidases produced by each species with Peptostreptococcus magnus and Peptostreptococcus micros belonging to group I, Peptostreptococcus asaccharolyticus and Peptostreptococcus prevotii belonging to group II, and Peptostreptococcus anaerobius forming group III. An analysis of possible proteolytic activity of four selected Peptostreptococcus magnus isolates indicated that only 5 of 11 substrates were hydrolyzed as compared to a control isolate of Porphyromonas gingivalis W83, which had a strong proteolytic profile. Therefore, gelatin hydrolysis by Peptostreptococcus spp., in particular Peptostreptococcus magnus and Peptostreptococcus micros, is probably due to a variety of aminopeptidases rather than proteinases.