The aim of the present study was to investigate the effects of a PGF 2α analogue (Dinoprost) on involution of the puerperal uterus, especially with pyometra during 35 to 45 days postpartum in multiparous high yielding Holstein dairy cows. At days 40 ± 5 postpartum, 1250 cows were ultrasonographically examined. Fifty cows were diagnosed as pyometric. Fifty pyometric and 50 clinically healthy cows were assigned randomly to one of four groups. Groups were: 1.) Pyometric+Dinoprost cows (PD, n = 25 treated with 25 mg of Dinoprost), 2.) Pyometric+Saline cows (PS, n = 25 treated with saline), 3.) Healthy+Dinoprost cows (HD, n = 25 treated with 25 mg of Dinoprost), and 4.) Healthy+Saline cows (HS, n = 25 treated with saline). All treatments were given intramuscularly. Ultrasonography was performed at the time of examination and 7 days later to evaluate changes in uterine diameter. Treatment with Dinoprost significantly reduced gross uterine diameter in PD in comparison to the PS group in the presence or absence (P4 concentrations <1 ng/ml) of an active corpus luteum. Dinoprost treatment did not decrease gross uterine diameter in groups HD and HS. Luminal diameter was significantly smaller in the PD than in the PS group. Pregnancy rate of PD was greater than PS cows (36% vs 20%, respectively). Days to first service (110 d vs. 140 d) and open days (160 d vs. 190 d) were shorter in PD cows than PS cows. It was concluded that injection of 25 mg of Dinoprost to pyometric cows had uterotonic effects in presence or absence of functional corpus luteum. Therefore, this treatment can help uterine involution in puerperal dairy cows affected by pyometra and consequently improved reproductive performance.