Use of antibiotics in subtherapeutic doses as growth-promoting feed additives for animal production is widespread in the U.S. and throughout the world. Previous studies by our research group concluded that size fractionation of poultry (broiler) litter followed by storage facilitated reutilization of litter as a soil amendment or bedding supplement. However, litter microbial contamination, including antibiotic-resistant populations, and accumulation of metals and other elements may limit litter reutilization. Litter from four broiler houses was separated into a fine fraction for use as a soil amendment, and a coarse fraction for reutilization as a bedding supplement in growing subsequent flocks of broilers. Fractions and whole litter were stored in indoor piles simulating farm storage conditions for 4 mo with periodic analysis for metals, other elements, and culturable bacteria (including total and fecal coliform, Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, and Campylobacter jejuni). Representative bacterial isolates were tested for their sensitivity to 12 common antibiotics (ampicillin, bacitracin, cephalothin, erythromycin, gentamicin, kanamycin, nalidixic acid, neomycin, penicillin, streptomycin, sulfisoxazole, and tetracycline) using the Kirby-Bauer technique. Pathogens and indicator bacteria tested were found to be resistant to multiple antibiotics. Data suggest that microbial contamination of litter should be reduced or eliminated prior to reutilization to minimize environmental health risks related to transfer of antibiotic-resistant bacteria to humans or other animals.