Our objectives were to test the efficacy of PGF_2α (PGF) to induce luteolysis and evaluate progesterone (P4) dynamics after intravaginal (IVG) administration in lactating dairy cows. Our hypothesis is that PGF given IVG will induce luteolysis. In 2 experiments, cows were synchronized using Ovsynch (GnRH-7 d-PGF-56 h-GnRH) to induce ovulation and the formation of a corpus luteum (CL). Cows with at least 1 CL ≥ 15 mm 8 d after Ovsynch remained in the study. In Exp 1, cows (n = 31) stratified by parity and number of CL received: 5 mL of saline solution IVG (SAL-IVG, n = 6), 25 mg of PGF im (PGF25-im, n = 7), 25 mg of PGF IVG (PGF25-IVG, n = 6), 50 mg of PGF IVG (PGF50-IVG, n = 6) and 125 mg of PGF IVG (PGF125-IVG, n = 6). In Exp 2, cows (n = 32) stratified as in Exp 1 received: SAL-IVG (n = 7), PGF25-im (n = 7), PGF25-IVG (n = 6), PGF50-IVG (n = 6) as in Exp 1 whereas another group received 2 IVG boluses of 25 mg of PGF 12 h apart (PGF25–2X-IVG, n = 6). Blood was collected at −1 h, 0 h, every 6 h up to 24 h, and then every 12 h up to 96 h after treatment (trt). Concentrations of P4 after treatment were analyzed by ANOVA with repeated measures using PROC MIXED of SAS. Mean separation was conducted with LSD method. In Exp 1, P4 concentrations from −1 to 96 h were affected by trt (P < 0.01), time (P < 0.001) and trt by time (P < 0.001). From 12 to 96 h, all PGF groups had less P4 (P < 0.05) than SAL-IVG cows. In Exp 2, from −1 to 96 h P4 concentrations were affected by trt (P < 0.001), time (P < 0.001) and trt by time (P < 0.001). From 12 to 96 h all PGF groups had less (P < 0.05) P4 than SAL-IVG cows. From 60 to 96 h, PGF25–2X-IVG and PGF25-im tended (P < 0.1) to or had less (P < 0.05) P4 than PGF25-IVG. When data from Exp 1 and 2 were combined, P4 concentrations from −1 to 96 h were affected by trt (P < 0.001), time (P < 0.001) and trt by time (P < 0.001). Concentrations of P4 in all the PGF groups were less (P < 0.05) than in the SAL-IVG group from 6 to 96 h. At 60 h, the PGF25–2X-IVG group had less (P < 0.05) P4 than the rest of the groups. We conclude that it is possible to induce luteolysis in lactating dairy cows by intravaginal administration of PGF. Two 25-mg boluses 12 h apart were more effective than one bolus of 25, 50 or 125 mg of PGF and as effective as a 25-mg bolus of PGF im.