In the ewe, the inhibition of pulsatile LH secretion by oestradiol during long days depends on dopaminergic activity and could involve amino acid transmitters. In the first experiment of the present study we observed the changes in LH secretion in ovariectomised ewes under long days immediately after subcutaneous implantation of oestradiol (peripheral treatment). In the second experiment, in order to identify the site of action of oestradiol, we observed the LH changes following intracerebral infusion of oestradiol through a microdialysis membrane (central treatment) within the preoptic area, the mediobasal hypothalamus (MBH) or the retrochiasmatic area (RCh) and measured amino acids and catecholaminergic transmitters and metabolites within the dialysates. With peripheral treatment, the amplitude, the nadir and the area under the LH pulse curve decreased within 4 to 8 h of the insertion of a subcutaneous oestradiol implant. After 18 h, the amplitude and the area under the pulses increased, as well as the intervals between pulses (from 49·9 ± 1·4 min to 75·6 ± 5·9 min). With central oestradiol treatment, LH changes were similar whatever the site of oestradiol infusion, suggesting either multiple sites of action or diffusion between structures. Twenty hours after the beginning of intracerebral oestradiol treatment, the amplitude and the area under the pulses increased, as did the interval between LH pulses (from 49·5 ± 4·1 min to 73·2 ± 14·2 min). Comparison of peripheral with central oestradiol treatment suggested that the long-lasting decrease in the nadir, as well as the transitory decrease in the amplitude and area, before 18 h in experiment 1 are reflections of hypophysial effects. In contrast, the increases in amplitude and area under the LH pulse curve seen 18–20 h after oestradiol in the two experiments could be due to the higher amplitude of LHRH pulses, as a result of an early stimulatory effect of oestradiol. After central oestradiol infusion, there was a decline in the concentration in the dialysate of two metabolites of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the RCh, suggesting an early inhibition of monoamine oxidase by the steroid. During the inhibition of LH pulsatility the concentration of γ-aminobutyric acid in the dialysate from the RCh and the MBH increased, suggesting the participation of this transmitter in the changes induced by oestradiol under long days.