Estrone sulfate is strongly bound by human serum albumin with one strong binding site (n1 ∼ 1) and several weaker binding sites. The apparent intrinsic association constants, k1 and k2, decrease with increasing concentration of albumin from 0.1–4.6% albumin. Serum albumin appears to be the only protein that binds estrone sulfate in human plasma. Estradiol-3-sulfate, estradiol-17-sulfate, estradiol-3,17-disulfate, estriol-3-sulfate, androsterone sulfate, and dehydropiandrosterone sulfate were shown to compete for estrone sulfate binding. Under physiological conditions, however, their concentrations are too low to measurably affect estrone sulfate binding in plasma. At 37 C, about 98.4% of endogenous estrone sulfate is bound in plasma of young adults.