The objective of this study was to assess the intrafollicular milieus across different follicle sizes and the associated outcomes of oocyte retrieval. Serum levels of estradiol and progesterone were measured on the day of oocyte retrieval in patients who underwent in vitro fertilization. Gene expression of Kiss-1, GnRH, PACAP, and their corresponding receptors within the cellular components obtained from follicles of different sizes were determined. Larger follicles contained higher levels of sex steroids compared with those in small follicles. The oocyte retrieval rate from the two different follicle sizes were identical. Intrafollicular sex steroid levels were not associated with the success of oocyte retrieval. Expression levels of Kiss-1, GnRH, and their receptor genes as well as PACAP gene expression in follicular cells of different sizes were identical, but gene expression of PACAP receptor was significantly lower in the cellular component of large compared with that of small follicles.