In an attempt to establish a defined model system for studies aimed at elucidating the mechanism of PGF2α action, we examined the effects of medium supplementation with soluble hydroxycholesterol analogues, alone and in combination with ovine luteinizing hormone (oLH) in the presence and absence of PGF2α' on progesterone secretion by mixed ovine luteal cells in vitro. In short-term cultures (2-6 h), supplementary 22R-hydroxycholesterol (22R-OHC; 0.16–20 μg ml−1) increased (P < 0.05) progesterone production in a dosedependent manner, whereas similar concentrations of 22S-hydroxycholesterol (22S-OHC) and 25-hydroxycholesterol (25-OHC) had little effect. In incubations of ≤24 h duration, 22R-OHC (1 μg ml−1) dramatically increased progesterone secretion, whereas oLH (100 ng ml−1) in the presence or absence of PGF2α (250 ng ml−1) had no consistent effects, alone or in combination with 22R-OHC. In contrast, 22R-OHC (1 μg ml−1) alone had no effect in long-term incubations (72–192 h), nor did treatment with oLH (100 ng ml−1) in the presence or absence of PGF2α (250 ng ml−1) in the absence of 22R-OHC. Together, however, 22R-OHC and oLH stimulated (P < 0.05) progesterone secretion, a synergistic effect consistently inhibited (P < 0.05) by PGF2α' Equimolar (2.5 μmol l−1) concentrations of 22R-OHC and homologous serum low- or high-density lipoprotein cholesterol exhibited comparable capacities to maintain progesterone secretion in long-term cultures (24–168 h), with and without gonadotrophin (oLH or human chorionic gonadotrophin, 100 ng ml−1) stimulation. These data establish 22R-OHC as an effective steroidogenic substrate for progesterone biosynthesis in ovine luteal cells in vitro, exhibit its capacity (when combined with gonadotrophin) to extend the useful life of cultured ovine luteal cells in a manner similar to serum lipoproteins and demonstrate the ability to reproduce PGF2α-induced luteolytic actions in a defined model system in vitro.