The objective of this study was to examine the effects of one or 2 doses of prostaglandin F_2α (PG) on progesterone (P₄) concentration profiles, and luteal function in dairy cows subjected to a 5-d CIDR-Cosynch protocol. On d 0, 43 cows received GnRH (100µg; i.m.), and a CIDR was inserted. On d 5, the CIDR was removed and cows were assigned randomly to receive one dose of PG (25mg) (1PG; n = 15), or 2 doses (25mg/dose) of PG 12 h apart (2PG; n = 19). On d 5 and after CIDR removal, serial blood samples were collected every 12 h from d 5 to 8 to measure P₄ concentrations. On d 8, all cows received a second GnRH injection and were inseminated. Ovaries were examined by ultrasonography on d 0, 5 and 8, and ovarian structures were recorded. Nine cows were determined acyclic and/or did not have luteal tissue at CIDR removal, thus were removed from this study. A repeated measures, generalized linear mixed model ANOVA was used to determine differences in P₄ concentrations. The model included treatment, the repeated factor time, and time by treatment interaction as fixed effects, and cow within treatment was the random effect. The P₄ data were assumed to follow a lognormal distribution. On d 5, all cows had a CL or developed luteal tissue, and P₄ concentrations were similar between groups (P > 0.1) before treatments. As expected, P₄ concentrations decreased over time in both treatments (P < 0.01). However, there was a significant effect of treatment by time interaction on P₄ concentrations (P < 0.01). By 24 h after treatments, P₄ concentrations were lower (P < 0.01), and remained lower (P < 0.01) throughout the the remainder of the experiment in 2PG compared with 1PG. By 72 h post-treatment, the average P₄ concentrations were 1.03 ng/mL and 0.05 ng/mL for 1PG and 2PG, respectively. These results indicate differences in P₄ repsonse between 1PG and 2PG in cows subjected to a 5-d CIDR-Cosynch. Compared with 1PG, 2PG injections (12 h apart) was more effective in inducing luteolysis, as P₄ concentrations were significantly less by the time of AI.