Bacillus subtilis, a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (delta(18)O) and hydrogen (deltaD) stable isotope ratios. Logarithmically growing cells and spores were each harvested from the cultures and their delta(18)O and deltaD values determined. Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water. We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B. subtilis. We then predicted the delta(18)O and deltaD values of spores produced in nutritionally identical media and local water sources for five different locations around the United States. Each of the measured delta(18)O and deltaD values of the spores matched the predicted values within a 95% confidence interval, indicating that stable isotope ratio analyses may be a powerful tool for tracing the geographic point-of-origin for microbial products.