Steroid hormones can act as potent endocrine disruptors when released into the environment. The main sources of these chemicals are thought to be wastewater treatment plant discharges and waste from animal feeding operations. While these compounds have frequently been found in wastewater effluents, few studies have investigated biosolids or manure, which are routinely land applied, as potential sources. This study assessed the relative environmental contribution of steroid hormones from biosolids and chicken litter. Samples of limed biosolids collected over a four year period and chicken litter from 12 mid-Atlantic farms were analyzed for 17β-estradiol (E2), estrone (E1), estriol (E3), 17α-ethinylestradiol (EE2), progesterone, and testosterone, and the conjugated hormones E1-sulfate (E1-S), E2-3-sulfate (E2-3-S), and E2-17-sulfate (E2-17-3). Results showed that E1 and progesterone were the most prevalent compounds in both of these materials, with E1-S also present in chicken litter.