Abstract Objective: To determine whether hCG and IGFBP-1 appear in the same or different cells and in what sequence. Design: Retrospective analysis of laboratory data. Setting: University medical center. Patient(s): Twenty-five women undergoing IVF-ET with natural cycles and 25 women having stimulated IVF-ET. Intervention(s): Cells were obtained from dominant follicles in women with natural cycles and from the follicles from hMG- and hCG-stimulated cycles. Main Outcome Measure(s): Detection and localization of hCG and IGFBP-1 in granulosa-luteal cells using double immunocytochemical staining. Measurement of hCG and IGFBP-1 in follicular fluid and serum. Result(s): Three types of hCG staining were found: on the cell surface, on the cell surface and in the cytoplasm, and in the cytoplasm alone. IGFBP-1 stained diffusely in the cytoplasm and was found only in those cells that were luteinized and contained hCG. IGFBP-1 and hCG were colocalized in the same cells. There was a positive correlation between follicular fluid hCG and IGFBP-1 levels, but only in natural IVF-ET cycles. Conclusion(s): HCG-driven luteinization is required for IGFBP-1 synthesis to take place in granulosa cells.