Thirteen strains of a novel spore-forming, Gram-positive, mesophilic heterotrophic bacterium were isolated from spacecraft surfaces (Mars Odyssey Orbiter) and assembly-facility surfaces at the Jet Propulsion Laboratory in California and the Kennedy Space Center in Florida. Phylogenetic analysis of 16S rRNA gene sequences has placed these novel isolates within the genus Bacillus, the greatest sequence similarity (99.9 %) being found with Bacillus pumilus. However, these isolates share a mere 91.2 % gyrB sequence similarity with Bacillus pumilus, rendering their 16S rRNA gene-derived relatedness suspect. Furthermore, DNA-DNA hybridization showed only 54-66 % DNA relatedness between the novel isolates and strains of B. pumilus. rep-PCR fingerprinting and previously reported matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protein profiling clearly distinguished these isolates from B. pumilus. Phenotypic analyses also showed some differentiation between the two genotypic groups, although the fatty acid compositions were almost identical. The polyphasic taxonomic studies revealed distinct clustering of the tested strains into two distinct species. On the basis of phenotypic characteristics and the results of phylogenetic analyses of 16S rRNA and gyrB gene sequences, repetitive element primer-PCR fingerprinting and DNA-DNA hybridization, the 13 isolates represent a novel species of the genus Bacillus, for which the name Bacillus safensis sp. nov. is proposed. The type strain is FO-36b(T) (=ATCC BAA-1126(T)=NBRC 100820(T)).