The binding affinity of various substituted estrogens for human sex steroid binding protein (SBP) and rat alpha-fetoprotein (AFP) have been measured by hydroxylapatite adsorption (relative to estradiol = 100%). While 17 alpha-ethynyl and 11 beta-methoxy substituents reduce the affinity of estrogens for these serum binding proteins markedly, a 16 alpha-bromo or a 16 alpha-iodo substituent actually increases their affinity for AFP, though lowering it for SBP. As a consequence, the uterine uptake selectivity of 16 alpha [77Br]-bromoestradiol (relative affinity for AFP = 230%) and 16 alpha [125 I]-iodoestradiol (relative affinity for AFP = 180%) in young rats (day 19-23), when AFP levels are still substantial, is considerably less than in older animals (day 24-27). 11 beta-Methoxy-16 alpha [77Br]-bromoestradiol, which has lower affinity for AFP (5.1%), does not show this age-dependent uptake selectivity. In adult cycling female rats bearing dimethylbenz(a)anthracene(DMBA)-induced mammary tumors, there is a strong dependence of uterine and tumor uptake selectivity on the stage of the estrous cycle: uptake is maximal during diestrus and minimal during estrus. The effective use of estrogen radiopharmaceuticals as receptor-based imaging agents requires careful consideration of not only the binding affinity of the agent for the estrogen receptor, but also its interaction with non-receptor binding proteins. The modulation of receptor concentrations by endogenous ligands during endocrine cycles and physiological differences between animals will also affect markedly certain measures of the extent of receptor-mediated uptake by target sites.