A variety of naturally occurring steroid hormones are regularly excreted by livestock, while additional steroid hormones have been used as growth promoters by the livestock industry. Depending on manure age and storage conditions, both groups of compounds are likely to be present during application to crops. Recent research suggests that some estrogens, androgens and progestagens in surface waters may originate from runoff after land application of livestock manure. Groundwater may also be impacted by livestock manure when used as a nutrient source to crops and may be indicated by excess nitrate in water. Few studies have been conducted to investigate the potential of steroid hormones contamination of groundwater. The objective of this study was to monitor leaching of steroid hormones and other compounds associated with livestock manure through the soil profile after land application of manure. The study was conducted near North Platte, Nebraska between April 2008 and July 2011 on a silt loam soil. Leachate was collected at the bottom of 2.4 meter deep monolithic percolation lysimeters to sample water leached beneath plots fertilized with manure. Soil samples were also collected from surrounding irrigated field plots. Treatments consisted of two manure handling procedures (stockpiling and composting) and a check receiving no manure application. Manure stored from a previous year’s cattle feeding pen study was sampled and analyzed for steroid hormone content. Manure was applied to the lysimeters and adjacent plot areas in April 2008 at a rate satisfying the nitrogen requirements of winter wheat planted in the fall of 2007 and 2008 followed by soybeans planted in the spring of 2010 and 2011. Leachate from the lysimeters and soil samples (down to 2.4 meter depth) from surrounding areas were collected periodically during the study. Laboratory analyses of manure, soil, and leachate samples used liquid chromatography tandem mass spectrometry to identify 17 steroid hormones and metabolites. Progesterone, estrone, beta-zearalenol and 4-androstenedione were detected at varying concentrations in both composted (1.6-8.4 nanograms per gram) and stockpiled (3.7-11.4 nanograms per gram) manure. Steroid hormones and related compounds were detected in only 5 percent of the leachate samples. The greatest detected concentration was 20 nanograms per liter of natural progesterone in a leachate sample from a lysimeter treated with stockpiled manure. Steroid hormones or metabolites were detected in 10 percent of the soil samples. Seventy four percent of the detections in the soil samples were in the top half (top 1.2 meter) of the sampled soil depth. 17beta-estradiol was detected the most in the soil samples (4 percent) with a maximum concentration of 4.3 nanograms per gram in a plot treated with composted manure. No synthetic steroids were detected in any of the soil or leachate samples. The low detection of steroid hormones in the soil and leachate samples suggests that, while some hormones may move through the soil, most are readily degraded or adsorbed after manure application. Additional research is required to more clearly identify the mechanisms that control the environmental fate and transport of steroid hormones through the soil