Estrogen and progesterone receptors prepared from mouse, rat, and human uteri, as well as from human breast cancers, have been characterized by gel-exclusion high-performance liquid chromatography. The qualitative relationships previously established by sedimentation analysis between the cytoplasmic [aggregated (approximately 8S), deaggregated (approximately 4S), and trypsinized (approximately 3.6S)] and nuclear (approximately 5S) forms of the rat uterine estrogen receptor were maintained by this technique. Differences in the partition of estrogen and progesterone receptors from the same species as well as interspecies differences in these receptors were reproducibly observed. Multiple forms of human estrogen and progesterone receptors could clearly be resolved in a single analysis and were distinct from serum steroid binding tissue contaminants. Separation analyses, performed at flow rates up to 2 mL min-1, were capable of resolving all receptor forms in 10--12 min with the column returning to base line in 25 min. With this exclusion gel column (TSK-G3000SW) as a background upon which to reference different receptor forms, eight distinct partitions or elution positions have been enumerated. This approach has considerable promise for the rapid characterization of different forms of steroid-receptor proteins. Moreover, it should provide a critical advantage in minimizing the opportunities for receptor modification during separation analysis and in maximizing the opportunity to study short-lived interactions between receptors and physiologic or pharmacologic ligands.