Food-borne illnesses have rarely been associated with the reuse of poultry litter as an organic fertilizer and soil amendment in agriculture. Yet farming practices in many countries have come under increased scrutiny because of heightened consumer awareness of food safety and environmental issues. This study was conducted to determine whether simple on-farm management practices could improve the microbiological safety of poultry litter. First, the effects of heat and moisture on the survival of Escherichia coli and Salmonella enterica serovar Typhimurium in poultry litter were investigated under laboratory conditions. Second, the persistence and regrowth of enteric bacteria were examined in poultry litter that had been aged for up to 12 wk in either a turned or static (unturned) windrow. Escherichia coli and Salmonella counts in poultry litter were reduced by >99% in 1 h at 55 or 65°C under laboratory conditions. At 35°C, both persisted longer under moist (65% wt/wt, wet basis) than dry (30% wt/wt) conditions. Poultry litter aged for 3 wk in a turned windrow, and up to 6 wk in a static windrow, supported increased E. coli densities when incubated in the laboratory at 37°C for 21 d. Peak temperatures >65°C were observed in both windrows within the first 3 wk of aging; after this point, the turned windrow was more consistently exposed to temperatures >45°C than the static windrow. By 12 wk, however, E. coli counts were very similar (3 to 3.6 log(10)) in the outside edge of both windrows. This study highlights the need for a better understanding of the interrelationship between spontaneous heating in organic waste streams, organic matter stabilization, and pathogen reduction.