To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential. Staining by four dyes and autofluorescence of B. subtilis spores that lack some (cotE, gerE) or most (cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC(6)(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC(6)(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC(6)(3) bound well to all germinated spores. The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate. The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.