Down-regulation of hCG receptors and a steroidogenic lesion in progesterone production were observed in isolated luteal cells after 24-h treatment of pseudopregnant rats with 50 IU hCG. The luteal cells from hCG-desensitized rats exhibited a complete loss of steroidogenic response to lipoproteins in both the absence and presence of hCG. This loss of response was not overcome by the addition of 25-hydroxycholesterol. To examine if the loss of steroidogenic response is due to a transient loss of steroidogenic enzymes or inadequate delivery of substrate cholesterol, cholesterol side-chain cleavage enzyme (P450scc) activity was measured in the isolated mitochondria. No differences appeared in enzyme activity between control and hCG-treated groups. Furthermore, electron paramagnetic resonance spectra of mitochondrial cytochrome P450scc from control and hCG-treated groups exhibited no appreciable differences in low spin electron paramagnetic resonance signal (g = 1.9, 2.2, and 2.4) or high spin EPR signal (g = 8.2), suggesting that the binding of the substrate to P450scc was not impaired. Additionally, the iron sulfur protein signal (g = 1.95 and 2.03) of the reduced mitochondria remained unchanged in the desensitized group. These data were supported by immunoblotting analysis which revealed no difference in the relative amounts of iron sulfur protein or cytochrome P450scc between the two groups. Moreover, the inner mitochondrial membranes, the locus of cytochrome P450scc, showed an identical cholesterol content in control and desensitized mitochondria, indicating that the availability of cholesterol substrate is not impaired during desensitization. These results suggest that the intramitochondrial transfer of cholesterol and cholesterol side-chain cleavage enzyme activity are unaffected in the desensitized state, but an inhibitory substance might be responsible for the lack of steroidogenic response.