We have shown previously that corpus luteum cells isolated from the superovulated ovaries of rats treated with 4-amino-pyrazolo[3,4-d]pyrimidine constitute a suitable experimental system by which to investigate the mechanism in which plasma high-density lipoprotein (HDL) plays a role in luteal cellular progesterone synthesis. In the present study, the rate of luteal cellular progesterone synthesis was shown to be stimulated by '"I-labelled HDL up to about 70% of the rate achieved in the presence of native HDL. The concentration of HDL needed for half-maximal stimulation of progesterone synthesis in the presence of lutropin was not significantly different irrespective of whether radioiodinated HDL or unlabelled HDL was used. Experimental conditions for studying the binding of 1251-labelled HDL to isolated luteal cells have been defined and cellular binding affinity and binding capacity have been measured. Exposure of the luteal cells to pronase virtually abolished their capacity to bind lZ51-HDL and made them unable to respond to added HDL by increasing their rate of progesterone synthesis in the presence of lutropin. Control experiments showed this effect of pronase on cellular progesterone synthesis not to be due to destruction of cellular lutropin receptors, nor to general cellular damage. This evidence supports the view that luteal cellular binding of HDL is part of the mechanism by which HDL acts in luteal progesterone synthesis. Cellular binding capacity and affinity for 1251-labelled HDL were the same irrespective of whether or not lutropin was present during incubation. Furthermore, the binding capacity and affinity of cells from the ovaries of rats not treated with 4-amino-pyrazolo[3,4-d]pyrimidine were the same as in luteal cells isolated from rats that had been treated.The work described in this paper is relevant to the mechanism by which high-density lipoprotein (HDL) acts in progesterone synthesis by the corpus luteum of the rat ovary. Using a preparation of isolated rat corpus luteum cells [l], we have already obtained and presented evidence that HDL plays such a role [2]. Briefly, we found that progesterone synthesis was very much impaired in corpus luteum cells isolated from rats which had been treated beforehand with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), a treatment which essentially deprives their tissues of access to circulating lipoproteins [2]. Rat HDL, but not other rat plasma lipoproteins, when added to the cell incubation medium, restored progesterone synthesis by these cells to normal levels [2]. Progesterone synthesis by corpus luteum cells isolated from rats which had not been treated with 4-APP was not significantly affected by added HDL [2]. Since deprivation of access to lipoproteins in vivo caused a defect in corpus luteum cell progesterone synthesis which was reversible by HDL presented in vitra, we concluded that HDL is necessary for luteal progesterone synthesis in the rat [2].