A possible luteolytic interaction between prostaglandin F2 alpha (PGF2 alpha) and estradiol benzoate (E2B) in cattle was investigated by randomly assigning 20 heifers to one of four groups of a 2 x 2 factorially designed experiment. The treatments for the groups consisted of im administration of: 1) 200 micrograms of E2B, given twice daily on d 10, 11 and 12 of the estrous cycle plus 7 mg PGF2 alpha, given at a separate site, but concurrently with the last injection of E2B; 2) the vehicles for E2B (sesame oil) and PGF2 alpha (saline); 3) E2B and saline and 4) sesame oil and PGF2 alpha. Administration of both E2B and 7 mg PGF2 alpha resulted in luteolysis as evidenced by a shorter mean length of the estrous cycle (P less than .05) when compared with the vehicle-treated control group, and a decline in systemic concentrations of progesterone to less than 1 ng/ml in four of five animals. Administration of PGF2 alpha with sesame oil was luteolytic in only one of five animals and E2B plus saline had no effect on the mean length of the estrous cycle or systemic concentrations of plasma progesterone. These observations suggested a luteolytic interaction between E2B and PGF2 alpha. This luteolytic interaction was examined further in a second experiment. Corpora lutea was removed 12 h after treatment with 200 micrograms E2B or .5 ml sesame oil, administered twice on d 10, 11 and 12 of the cycle, and incubated with luteinizing hormone (LH) and PGF2 alpha in vitro. The effect of both LH and PGF2 alpha was to increase progesterone synthesis (P less than .01), and this effect was observed irrespective of E2B pretreatment. These findings were not consistent with the hypothesis that the luteolytic site of action for either E2B or PGF2 alpha was inhibition of the steroidogenic effects of LH as assessed under in vitro conditions.