The objective of this study was to further test the hypothesis that follicular secretions produced by follicular cells with different maturity affect oocyte cytoplasmic maturation differently. A group of nine gilts were slaughtered on Day 17 of the estrous cycle and another group of ten were slaughtered on Day 20. The five largest and five smallest follicles were selected from the fifteen largest healthy follicles dissected from each pair of ovaries and categorized as large and small follicles, respectively. The five follicular shells in each category were pooled and cultured in 10 ml of modified TCM 199 supplemented with 2.5 mu g FSH/ml for 48 h with use of a mobile culture system. Afterwards, conditioned media were collected and used for maturing immature porcine oocytes. Each of six replicated experiments consisted of two culture dishes for each conditioned medium: Day 17 small (S17) and Day 17 large (L17), and Day 20 small (S20) and Day 20 large (L20) follicular shells. After 47 h culture, oocytes were inseminated with boar spermatozoa and were fixed 11 h post-insemination to examine fertilization. The average diameters of follicles used were 4.4, 5.7, 6.2, and 7.5 mm for S17, L17, S20, and L20, respectively. Nuclear maturation was slightly higher in oocytes matured in conditioned media produced by large follicular shells than in those produced by small follicular shells (97.0 vs. 93.5%, P = 0.045). Penetrability of oocytes was not affected by treatments. Male pronuclear formation in penetrated oocytes was, however, significantly affected by follicular age (Day 17: 17.4% vs. Day 20: 19.4%, P = 0.009), but not by follicular size (small: 25.9% vs. large: 30.9%, P = 0.363). These results confirm that diversity of follicular development in naturally cyclic gilts may affect the quality of oocytes ovulated.