The present study was designed to establish appropriate conditions for cell cycle analysis using flow cytometry and to characterize the cell cycles of granulosa cell populations during follicular maturation in cattle. Bovine ovaries were transported from a local abattoir to laboratory in saline. From morphologically normal follicle, follicular fluid was aspirated using 10 ml syringe with 21 gauge needle and passed through a 200 μm steel filter (Nalgene, USA) to remove large pieces of tissues or cell clumps. Granulosa cells isolated by Percol gradient were grouped to follicular size as follows: F1 (>10mm), F2 (5 to 10mm), F3 (3 to 4mm) and F4 (1 to 2mm). These cells were fixed and stained with propidium iodide. Flow cytometer (FACStar p l u s , Becton Dickinson Immunocytometry System, USA) equipped with a laser excitation system was used to examine the relative cell size and granularity of cells by method described by Rao et al. (Anat. Rec. 229:177-185) and to analyze the stage of cell cycle of 5,000 individual cells in each group by previously reported method (Marrone and Crissman, Endocrinology 122:651-685). In forward angle light-scatter and 90° light-scatter analysis, granulosa cell populations (gap 1 phase (G 1 ) of cell cycle) from each follicle were relatively regular in size and granularity, regardless of follicular size. However, their distribution in granularity was greater than that in size. Most of granulosa cells collected from each follicle were distributed in gap 0 (G 0 )/G 1 , synthesis (S) and G 2 /M (mitosis) phases (Table). As the follicles approached ovulation, the percentage of cells in the proliferative phases of cell cycle (S and G 2 /M) decreased significantly, with a concomitant increase in the percentage of granulosa cells in G 1 phase. Therefore, our results indicate that the proportion of main populations to cell cycle of granulosa cells may be changed from proliferative phase to G 1 phase during follicular maturation in cattle.