The aim of this study was to examine the hypothesis that follicular and uterine perfusion as well as endometrial gene expression during the ovulatory period differs after induction of luteolysis during the 1st follicular wave compared with the 2nd wave or in intact cycle. Nine healthy non-lactating Holstein-Friesian cows were examined during three estrous cycles. A prostaglandin F2α analogue (PGF) was administered randomly either on Day 7 (1st wave cycle) or Day 11 (2nd wave cycle) after detection of ovulation (=Day 1). No hormonal treatment was used in the intact cycle with spontaneous ovulation. Transrectal Doppler sonography was conducted daily after PGF injection and in the intact cycle beginning on Day 18 of the estrous cycle until ovulation. Follicular blood flow (FBF) was determined by measuring the maximum area of colour pixels on digitalized images of the follicles. Uterine blood flow was quantified by the time-averaged maximum velocity (UTAMV) and pulsatility index (PI) in both uterine arteries. Blood flow measurements were carried out on Days -1 and 0. Endometrial biopsy specimens were taken on Day 1 and analyzed for the gene expressions of estrogen, progesterone, oxytocin and VEGF receptors and eNOS and iNOS using RT-PCR. The interval from PGF injection to ovulation was shorter (P<0.05) in 1st wave cycles than in 2nd wave cycles. On Days 0 and -1, FBF was greater (P<0.05) in 1st wave cycles than in intact and 2nd wave cycles. On Day -1, UTAMV was greater (P<0.05) in 1st wave cycles than in intact and 2nd wave cycles. There were no differences (P>0.05) in FBF and UTAMV values between 2nd wave and intact cycles. No differences (P>0.05) were detected in the gene expressions of endometrial receptors and enzymes between intact, 1st and 2nd wave cycles. The results show that follicular and uterine perfusion during the ovulatory phase are higher after induction of luteolysis during the 1st follicular wave compared with the 2nd wave or intact cycle. There were no effects on endometrial gene expression.