To assess whether the prokinetic effects of B. subtilis were due to the bacteria’s live nature as well as its Trp-synthesizing capability, motility was assessed following a week of treatment with either heat-inactivated B. subtilis R0179 or with the tryptophan synthase lacking strain B. subtilis 1A2 (B. sub Trp syn-). (A-C) Mice that received heat-inactivated B. subtilis R0179 (n=11) or B. subtilis 1A2 (n=12) did not exhibit changes in whole gut transit time after a week of treatment. These treatment groups were not significantly different from each other or vehicle (n=5) in percent change from baseline. (D-F) In addition to whole gut transit, there was no significant change in colonic motility times in mice treated with either heat-inactivated B. subtilis R0179 (n=12) or B. subtilis 1A2 (n=7). Furthermore, there were no differences between both bacteria treatments or vehicle (n=6). Data are shown as individual animals (A,B,D,E), paired Student’s t-test, or mean ± SEM (C,F), one-way ANOVA with Tukey’s post-hoc multiple comparisons test.