To develop an effective immunization protocol against human serum albumin-Cys-Gly-GnRH (HSA-GnRH) conjugate to delay the onset of puberty in heifers, 58 heifers (8 mo of age; mean +/- SE BW = 203 +/- 1 kg) were randomly assigned to each of six treatments: 1) controls, .1 mg of HSA, with diethylaminoethyl (DEAE)-dextran as adjuvant, on d 0 and 28; 2) .1 mg of HSA-GnRH, with DEAE-dextran, on d 0; 3) as 2) and booster on d 28; 4) as 3) but boosters also on d 84, 140, 196, and 252; 5) as 2) but half the conjugate given with DEAE-dextran adjuvant and half with non-ulcerative Freund's adjuvant (NUFA), injected in two separate sites; and 6) as 2) but the conjugate given with DEAE-dextran and NUFA, emulsified and injected in two sites. The duration of the experiment was 342 d. Mean plasma GnRH antibody titers (samples every 2 wk) for heifers in Treatments 2 to 6 were 9.4 +/- 1.16, 20.6 +/- 2.21, 43.9 +/- 2.86, 27.9 +/- 2.67, and 44.5 +/- 3.75% binding at a plasma dilution of 1:640. The mean number of times estrus was observed in heifers was less (P < .05; pooled SEM = .53) in Treatments 4 (.2) and 6 (2.4) than in Treatments 1, 2, 3, and 5 (7.8, 7.0, 7.0, and 6.6, respectively). The mean interval to the onset of puberty (the first increase in plasma progesterone > or = .5 ng/mL for > or = 10 d with samples at 3- to 4-d intervals) was greater (P < .05; pooled SEM = 11.6) for heifers in treatments 4 (339 d) and 6 (276 d) than for heifers in Treatments 1, 2, 3, and 5 (164, 159, 165, and 170 d, respectively). Mean ADG of heifers was reduced (P < .05) in treatments 2, 3, 4, and 6 (.71, .72, .68, and .69 kg, respectively) compared with controls (.77). In summary, the multiple booster immunization treatment induced and maintained sufficient anti-GnRH titer to delay puberty for 175 d; a single immunization against GnRH with DEAE and NUFA increased antibody titers enough to delay puberty for 112 d. However, GnRH immunization treatments reduced ADG of heifers in Treatments 2, 3, 4, and 6.