In gram-negative organisms, enzymes belonging to the low-molecular-weight protein tyrosine phosphatase (LMPTP) family are involved in the regulation of important physiological functions, including stress resistance and synthesis of the polysaccharide capsule. LMPTPs have been identified also in gram-positive bacteria, but their functions in these organisms are presently unknown. We cloned two putative LMPTPs from Bacillus subtilis, YfkJ and YwlE, which are highly similar to each other in primary structure as well as to LMPTPs from gram-negative bacteria. When purified from overexpressing Escherichia coli strains, both enzymes were able to dephosphorylate p-nitrophenyl-phosphate and phosphotyrosine-containing substrates in vitro but showed significant differences in kinetic parameters and sensitivity to inhibitors. Transcriptional analyses showed that yfkJ was transcribed at a low level throughout the growth cycle and underwent a sigma(B)-dependent transcriptional upregulation in response to ethanol stress. The transcription of ywlE was growth dependent but stress insensitive. Genomic deletion of each phosphatase-encoding gene led to a phenotype of reduced bacterial resistance to ethanol stress, which was more marked in the ywlE deletion strain. Our study suggests that YfkJ and YwlE play roles in B. subtilis stress resistance.