The objective of this experiment was to develop a procedure for immunizing ewes against melatonin that would alter the effects of changing photoperiod on seasonal reproduction and prolactin secretion. Ewes were immunized against human serum albumin (HSA) as controls (n = 9) or a melatonin-human serum albumin conjugate (0.25 mg; n = 10) on December 14th (Day 0) and boosted 9 times. They were maintained on natural photoperiod and then transferred indoors and exposed to long days for 35 d, followed by short days for 146 d, long days for 93 d, and short days for a further 123 d. Antibody titers to melatonin (at a serum dilution of 1:1,250) were significantly higher in immunized ewes (27.3 +/- 6.6%) than controls (0.7 +/- 0.1%; P < 0.001). At the end of the experiment, antibody titers in immunized ewes (at dilution of 1:50) were higher in blood (43.7 +/- 8.2%) than in cerebrospinal fluid (10.8 +/- 3.9%; P < 0.05), and highly correlated (r2 = 0.746). Onset of the breeding season was advanced slightly after the second transfer from long to short days in immunized ewes (April 12 +/- 3 d) compared with controls (April 25 +/- 3 d; P < 0.05). Mean serum prolactin concentrations were lower (P < 0.05) in melatonin-immunized ewes compared with controls on natural photoperiod, after transfer from long to short days, during long days, and after the second transfer from long to short days. In conclusion, despite melatonin-immunization increasing antibody titers in blood and cerebrospinal fluid, and decreasing prolactin concentrations over much of the experiment, minimal effects on the timing of reproductive transitions in the ewes were evident. This discrepancy between the response of the prolactin and reproductive axes to melatonin immunization supports the hypothesis of a dual site of action of melatonin, with melatonin acting in the pituitary gland to mediate the effects of photoperiod on prolactin secretion and in the mediobasal hypothalamus to affect reproductive responses.