Objective: To find the differences between human granulosa-luteal cells obtained during early timed follicular aspiration to prevent severe ovarian hyperstimulation syndrome (OHSS) and during normally timed follicular aspiration. Design: Retrospective analysis of clinical laboratory data. Setting: In vitro fertilization unit, University Department of Obstetrics and Gynecology, Ljubljana, Slovenia. Patient(s): Twenty women undergoing IVF-ET at high risk for OHSS. Intervention(s): Cells were obtained from the follicles of women who were stimulated with hMG and hCG during an early timed follicular aspiration of one ovary, 10–12 hours after hCG, and during a normally timed follicular aspiration of the contralateral ovary, 32–36 hours after hCG administration. Main Outcome Measure(s): Subpopulations of granulosa-luteal cells were observed by computerized image analysis in which hCG was localized using immunoperoxidase staining. Result(s): Early timed follicular aspirates contained no oocytes and only a scant number of granulosa cells. Granulosa-luteal cells were smaller than those from normally timed follicular aspirates. We identified three subpopulations in early timed follicular aspirates: nonluteinized, small luteinized, and medium luteinized cells. In normally timed follicular aspirates, four subpopulations were identified, including large luteinized cells. The normally timed follicular aspirates contained more hCG-stained cells. Three staining types of hCG localization were found: on the surface membrane, on the surface membrane and within the cytoplasm, and only within the cytoplasm of cells from normally timed follicular aspirates. Early timed follicular aspirates contained only cells with membrane hCG localization. Conclusion(s): We found differences in morphometric characteristics and hCG localization between human granulosa-luteal cells obtained during early timed follicular aspiration to prevent severe OHSS and during normally timed follicular aspiration.