To optimize the prostaglandin Fa, (PGF) immunization protocol (conjugate [PGF-human serum albumin; PGF-HSA] dose and immunization regimen) to achieve prolonged suppression of estrous behavior (EB) in beef heifers, 56, 14-mo-old cyclic heifers were assigned (n = 7 per treatment) to eight treatments: 1) 3.3 mg of PGF-HSA on d 0 (single); 2) 3.3 mg of PGF-HSA on d 0 and 28 (booster; B); 3) as (2) except on d 0 and 55; 4) as (2) except on d 0 and 83; and 5 to 8) as in Treatments 1 to 4 except using 10 mg of PGF-HSA. The adjuvant was diethylaminoethyl-dextran, and duration of the experiment was 170 d. Heifers were checked twice daily for EB. A persistent corpus luteum (CL) was considered present when progesterone (P-4) was greater than or equal to .5 ng/mL for 1 six consecutive samples (every 3 to 4 d). Data were analyzed using ANOVA for a factorial plan. All heifers produced plasma antibody titers (samples every 2 wk) against PGF (peak range: 7 to 84% binding at 1: 1,250). There were no effects (P > .10) of conjugate dose and no interactions between dose and immunization regimen for any variable; therefore, data were combined across dose. Mean and peak titers were greater (P < .05) in heifers in 55- and 83-d B treatments than those in single immunization and 28-d B treatments. Overall, 48/55 heifers formed a persistent CL (41/41 for B heifers). In the single, and 55- and 83-d B treatments, 23/42 heifers formed persistent CL in response to single/primary immunization. There was no difference between immunization regimens in duration (133 +/- 4.4 d) of persistent CL. In summary, dose of conjugate did not affect any variable; however, single immunizations were,less effective in forming persistent CL and longer B intervals were more effective in increasing antibody titers, but B interval did not affect the incidence of persistent CL.