Bacillus cereus GS1 secretes a diffusible bacteriocin-like molecule into culture medium that can be detected by a modified spot-on-lawn agar plate method. The secreted molecule exhibits a narrow inhibitory spectrum, affecting only the growth of Bacillus subtilis 6633 and certain other Gram-positive species. The molecule, in cell-free supernatant, is heat-stable and resistant to degradation by a number of proteases and to denaturation by several solvents. Clearing on agar plates is inversely related to the ionic strength of the growth medium, consistent with a membrane-directed activity. Analysis of activity gels reveals a clearing that coincides with a low molecular weight protein of about three kilodaltons. The inhibitory spectrum and protease sensitivity profile observed for this bacteriocin-like molecule distinguish it from similarly described molecules produced by other strains of Bacillus cereus. Our results support our hypothesis that the inhibitory molecule is a novel, low molecular weight bacteriocin from Bacillus cereus and we propose naming it Cerein GS1.