Fluorine-18 16alpha-Fluoroestradiol ([18F]-FES) is a positron-emitting tracer for the estrogen receptor that is used for positron emission tomography (PET) studies of tumor tissues rich in the estrogen receptor. The role of the sex steroid binding protein (SBP or SHBG) in the transport of the [18F]-FES to the estrogen-receptor-rich tissue in breast cancer patients in vivo was investigated. To determine the extent to which [18F]-FES is bound to SBP in the blood, we performed a series of studies using blood samples obtained from patients undergoing [18F]-FES PET scans. The binding of [18F]-FES to the SBP was measured using a simple protein precipitation assay. The binding of [18F]-FES metabolites to SBP was also measured. These measurements showed that the tracer was distributed between albumin and SBP, and the binding capacity of SBP was sufficient to ensure that the protein was not saturated when the tracer was fully mixed with the plasma; however, local saturation of SBP may occur when [18F]-FES is administered intravenously. Typically about 45% of [18F]-FES in circulating plasma was bound to SBP, but this fraction was dependent on the concentration of SBP in plasma. The transfer of the tracer between the two proteins was rapid, complete in less than 20 s at 0 degrees C, suggesting that the equilibrium was maintained under most circumstances and that local saturation resolved quickly when blood from the injection site entered the central circulation. These data suggest that SBP binding of [18F]-FES is significant and will affect the input function of the tracer for any model that is used for the quantitative evaluation of [18F]-FES uptake in PET studies. Estimates of equilibrium binding in blood samples are sufficient to characterize [18F]-FES binding to SBP in the circulation.