The objective of the work proposed is to characterize autoradiographically demonstrable sex steroid concentrating cells in the rat brain (I,A) by their neural connections, (I,B) histochemistry and (II) by the steroid specificity of the sex steroid binding. The locations of sex steroid concentrating neurons suggest involvement in control of neuroendocrine events and reproductive behavior. However, more facts on the cytologic and hormone-binding properties of individual cells are required to approach a direct cell-specific proof of their involvement in reproductive processes. We will use neuroanatomical, neurohistochemical, and steroid hormone autoradiographic techniques, and new combinations of these techniques, to provide cell-specific information on hormone-binding neurons. (I,A) The horseradish peroxidase (HRP) method will be used for the retrograde identification of hypothalamic neurons by their efferent connections. Then we will combine the HRP method with the specialized technique of steroid autoradiography to study the axonal projections of individual hypothalamic estradiol-concentrating neurons. (I,B) The tetrazolium formazan histochemical method will be used to localize individually (and map) neurons (in hypothalamic and limbic structures) containing six enzymes previously shown to be estradiol-sensitive. Then we will combine the tetrazolium formazan histochemical method with steroid hormone autoradiography to study enzyme activities in individual estradiol-concentrating neurons. (II) Quantitative use of sex steroid autoradiography will demonstrate receptor properties of individual sex steroid concentrating cells. We will do competition and cross-competition studies to determine the steroid specificity of hormone-binding neurons in hypothalamic and limbic cell groups, and the effects of known steroid antagonists will be analyzed. Characterizing sex steroid-binding neurons on a cell-by-cell basis in hypothalamic and limbic structures will point the way to direct physiologic and endocrine analyses of their precise role in reproductive functions.