Aerobic, endospore-forming bacteria that are attributed to the genus Bacillus or related genera constitute a hazard to the quality of gelatin. During repetitive extragenic palindromic DNA (rep)-PCR screening of gelatin isolates, a group of five isolates (group 1) and a group of 66 isolates (group 2) that did not match any pattern in our database were found. On the basis of 16S rDNA sequence analysis, representative strains of the different rep-PCR fingerprint types of group 1 were shown to be related most closely to Anoxybacillus species, but with sequence similarity of <97 %. Likewise, representative strains of group 2 were shown to be related most closely to Bacillus species, with 16S rDNA sequence similarity of <97 %. DNA-DNA reassociation values of isolates that displayed the most divergent rep-PCR profiles revealed that strains within each group belonged to a single species, according to recommendations for species delineation. A mean fatty acid profile could be calculated for each group. Isolates within a single group had similar patterns of results in API and other phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen. Physiological characterization of group 1 isolates allows their distinction from other Anoxybacillus species. Despite the weak reaction of group 2 isolates in API tests, physiological characterization allows distinction between Bacillus species that react weakly in API tests. Two novel species are therefore proposed, with the names Anoxybacillus contaminans sp. nov. (type strain, LMG 21881(T)=DSM 15866(T)) and Bacillus gelatini sp. nov. (type strain, LMG 21880(T)=DSM 15865(T)).