Inadvertent use of prostaglandin F2α (PGF2α) in pregnant cattle could result in luteolysis and pregnancy loss. Using a nonpregnant cow model, we determined if luteotropic agents could counteract luteolytic effects of PGF2α. Ovarian status of 20 lactating nonpregnant Holstein cows was synchronized using an Ovsynch protocol and ovulation confirmed by transrectal ultrasonography 48 hours after the second gonadotropin releasing hormone (GnRH). Eight days after ovulation, 25 mg of dinoprost (native PGF2α) was administered IM to induce luteolysis. Five minutes after PGF2α, cows were treated IM with gonadorelin (GnRH, 100 μg; n = 5), human chorionic gonadotropin (hCG, 1,000 IU; n = 6), porcine pituitary luteinizing hormone (pLH, 25 mg; n = 5), or saline (control, 2 ml; n = 4). Blood samples were collected before PGF2α (0 hour) and at 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72, and 84 hours after PGF2α to monitor subsequent luteal activity by measuring plasma progesterone concentrations. Although none of the treatments counteracted the luteolytic action of PGF2α, the rate of decline in plasma progesterone within 1 hour after PGF2α was greater (p = 0.04) in control than in GnRH, hCG, and pLH (2.2 versus 0.2, 0.3, and 0.1 ng/ml/hour, respectively). In addition, pLH-treated cows tended (p = 0.07) to have greater overall mean plasma progesterone for up to 84 hours (1.6 ± 0.2 ng/ml) than other treatments combined (1.1 ± 0.1 ng/ml). In the present study, giving GnRH, hCG or pLH 5 minutes after PGF2α administration was not effective in countering luteolytic effects of PGF2α. Nevertheless, pLH treatment tended to delay PGF2α induced luteolysis, which warrants further investigation.