The release of endocrine disrupting chemicals (EDCs) into the environment is of concern due to their potential deleterious effects on freshwater ecosystems and human health. Estrogen losses from agricultural land to freshwater have been implicated as a diffuse source of EDC pollution, however, uncertainty exists about the magnitude of this flux in comparison to other point sources (e.g. sewage treatment plants). Recent reviews have all highlighted the need for more mechanistic studies on hormone behaviour in soil environments. The aim of this study was to investigate the influence of aqueous matrix on the leaching, sorption and persistence of two naturally occurring hormones, estrone and 17β-estradiol in three agricultural grassland soils. The hormones were applied to the surface of the soil in two solvents, distilled water and natural sheep urine. Rainfall was subsequently applied to the top of the soil columns and the leachate collected. In comparison to distilled water, the presence of sheep urine both enhanced and prolonged the amount of estrogen leaching from soil. We hypothesized that this enhanced rate of estrogen migration in soil was due to changes in either estrogen sorption or microbial activity. While the presence of urine did not greatly affect the rate and amount of estrogen sorption to soil (Kd = 4-16) it did significantly reduce the rate of hormone mineralization. Overall, our study shows that vertical estrogen movement is rapid, soil type dependent and regulated by the aqueous matrix in which the hormones are contained. In terms of risk assessment and environmental fate modelling, we conclude that previous studies performed using hormones contained in artificial matrices (e.g. distilled water) may have underestimated their rate of dissipation in the environment.