Plasma membranes of ovarian luteal and adrenal cortical cells from "microvillar channels," a unique extracellular compartment formed by the close apposition of flattened microvillar surfaces. Microvillar channels have unusual affinity for cholesterol-rich lipoproteins, and, in vivo, may provide an increased surface area for these particles. In this research, we have isolated a plasma membrane-enriched fraction from rat luteinized ovaries, in which closely apposed membrane (i.e. microvillar channels) comprise about 30% of the preparation. Following in vitro incubations (approximately 1 h) of this plasma membrane fraction with different plasma lipoproteins, the closely apposed plasma membrane surfaces widen and become filled with lipoprotein particles (up to about 30 nm), whereas other membranes of the fraction show little binding. Competition experiments show that rat high density lipoproteins have the highest affinity for binding to the plasma membrane fraction. Radiolabeled plasma lipoprotein and the tissue-specific hormone, human chorionic gonadotropin, showed specific and saturable binding to the plasma membrane fraction, whereas other macromolecules used as controls did not. Radioautographic analyses of 125I-labeled lipoproteins and human chorionic gonadotropin indicate that binding occurs predominantly to the closely apposed plasma membranes (i.e. microvillar channels of the fraction). These studies show that microvillar channels of steroid-secreting cells entrap large numbers of plasma lipoproteins, particularly high density lipoproteins particles, presumably functioning in the delivery of cholesterol to these cells.