Research question: How is the production of progesterone (P₄) and 17-hydroxy-P₄ (17-OH-P₄) regulated between theca cells and granulosa cells during the follicular phase, during ovulation and after transformation into a corpus luteum? Design: Three cohorts were examined: (i) 31 women undergoing natural and stimulated cycles, with serum hormone measurements taken every 3 days; (ii) 50 women undergoing ovarian stimulation, with hormone concentrations in serum and follicular fluid assessed at five time points during final follicle maturation; and (iii) 12 women undergoing fertility preservation, with hormone concentrations evaluated via the follicular fluid of small antral follicles. Results: In the early follicular phase, theca cells primarily synthesized 17-OH-P₄ while granulosa cells produced limited P₄, maintaining the P₄:17-OH-P₄ ratio <1. As follicles reached follicle selection at a diameter of approximately 10 mm, P₄ synthesis in granulosa cells was up-regulated, but P₄ was mainly accumulated in follicular fluid. During final maturation, enhanced activity of the enzyme HSD3B2 in granulosa cells enhanced P₄ production, with the P₄:17-OH-P₄ ratio increasing to >1. The concentration of 17-OH-P₄ in the luteal phase was similar to that in the follicular phase, but P₄ production increased in the luteal phase, yielding a P₄:17-OH-P₄ ratio significantly >1. Conclusions: The P₄:17-OH-P₄ ratio reflects the activity of granulosa cells and theca cells during the follicular phase and following luteinization in the corpus luteum. Managing the function of granulosa cells is key for reducing the concentration of P₄ during ovarian stimulation, but the concerted action of FSH and LH on granulosa cells during the second half of the follicular phase makes this complex.