We have validated a hydroxylapatite assay for measuring estrogen receptor in extracts from breast tumor nuclei. By adsorption of receptor of hydroxylapatite prior to addition of radioactive ligand and warming, receptor degradation can be avoided. Total binding sites are measured at 30 degrees by exchange, and receptor sites unoccupied by steroid are measured at 4 degrees. A single saturating dose of 5 nM tritiated estradiol (with or without a 100-fold excess of nonradioactive diethylstilbestrol to estimate nonspecific binding) yields results similar to a six-dose Scatchard plot. Following in vivo injection of estradiol into rats bearing mammary tumors, receptor translocation in the tumors can be accurately quantitated with this assay. Applying the assay to human breast cancer, we find that tumor biopsies may contain cytoplasmic receptor alone or may also have appreciable nuclear receptor. The latter may be bound to estradiol or may be found in "free" form. The finding of free receptor in the nuclei in certain cases raises the possibility that unoccupied receptor might be able to stimulate cell replication in these cases, even in the absence of estrogen.