To examine the relationship between blood levels of 16 alpha-[18F]fluoro-17 beta-estradiol(18F-ES) and serum alphafetoprotein (AFP) concentration, we undertook a study in which serum from various aged (20-33 days old) Sprague-Dawley female rats injected with 18F-ES was analyzed for both blood activity levels and AFP. There is a strong positive correlation between serum AFP concentration and 18F-ES blood levels (r = 0.914, P less than 0.001), suggesting that the binding of 18F-ES by AFP has a significant effect on blood activity levels. The AFP concentration and ultimately the AFP-18F-ES binding is dependent on the age and weight of the rat: younger, as well as low weight rats exhibited high AFP concentrations and consequently increased 18F-ES blood activity. The rats most suitable for comparative studying of labeled estrogens are 25-28 days of age and weigh a minimum of 50-55 g. Thus, the use of the immature rat model to compare labeled estrogens requires a careful consideration of possible interference from blood binding proteins (i.e. AFP), as well as potential receptor binding competition from endogenous estrogens produced during the estrous cycle. Comparable consideration of blood binding proteins (sex steroid binding protein, SBP) and endogenous estrogens must be made in human studies, as well.