The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17α-hydroxylase, cytochrome P450 aromatase, 3β-hydroxysteroid dehydrogenase Δ⁴,Δ⁵ isomerase, LH, and FSH receptors and estrogen receptor-β in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17β, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17β (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17β concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-β mRNAs than other groups. In summary, increased expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17β concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.