The aims of this study were to determine the concentrations of the progesterone, oestradiol-17-β, vitamin A, C and β-carotene in plasma and cyst fluid and to relate these values with cystic diameter and membrane thickness of Holstein cattle with ovarian luteal cyst. 1650 Holstein cows were examined for the presence of the ovarian cyst and luteal and follicular cystic ovaries were obtained following slaughtering in personal slaughterhouse in Konya-Turkey. 15 Luteal and 15 follicular cystic ovaries were distinguished by rectal palpation and by post mortem ultrasonographic examination. Plasma and cyst fluid, hormone and vitamin analyses were carried out by EIA method and spectrophotometric measurement respectively. Although there was no relationship between β-carotene and vitamin A in plasma and cyst fluid of both cyst type and hormone concentrations, the vitamin C concentration of cyst fluid was found significantly higher in luteal cyst than in follicular cyst. Moreover, there is a positive correlation among values of the vitamin C concentrations of cyst fluid and cystic membrane thickness, plasma and the cyst fluid progesterone concentrations, but there is a negative correlation among the vitamin C concentrations of cystic fluid and oestradiol 17β levels of plasma and cyst fluid. In conclusion, vitamin C concentration of cyst fluid supported ultrasonographic and endocrinologic findings. Also, it can be postulated that vitamin C is probably effective on progesterone synthesis in the luteal tissue of cyst. © 2008 The Authors.