Nod1 and Nod2 are intracellular proteins that are involved in recognition of bacterial molecules and their genetic variations have been linked to several inflammatory diseases that are strongly affected by environmental factors. However, the distribution of Nod1- and Nod2-stimulatory molecules in different bacterial species and environments is unknown. Here we established a quantitative bioassay to screen and characterize Nod1- and Nod2-stimulatory activities in different environmental sites and bacterial species. Using this system, we found that common environments including foods and soils contain high levels of Nod1- and Nod2-stimulatory activities. Several Bacillus species were identified to possess the highest Nod1-stimulatory activity among soil bacteria. Unlike other immunostimulatory molecules, the higher level of Nod1-stimulatory activity was found in the culture supernatant and not in extracts from whole cell bacteria. Nod1-stimulatory molecules were highly stable at extreme pH and boiling conditions and were synthesized in an amidase- and sltY-independent manner. These results suggest a novel mechanism by which bacteria present in the environment stimulate the host immune system through Nod1.