Methionine-enkephalin (met-enk) levels were measured at 09.30–10.30, 16.30–17.30 and 23.30–00.30 h in the anterior hypothalamus-preoptic area (AHPOA), medial basal hypothalamus (MBH), anterior, intermediate and posterior lobe of the pituitary gland of male rats. Met-enk levels in the AHPOA and MBH fluctuated significantly through a 24-hour period; from low levels at 09.30–10.30 h, met-enk in the AHPOA and MBH rose slowly in the afternoon to reach peak concentrations at 23.30–00.30 h. Pinealectomy appears to depress or shift the normally occurring increments in met-enk levels at 23.30–00.30 h in the AHPOA only. Serum testosterone (T) levels displayed an inverse relationship with the met-enk rhythm; high concentrations during the day were associated with low met-enk levels and as T levels decreased to a nadir at 23.30–00.30 h, the met-enk in the MBH and AHPOA attained peak concentrations. Daily changes in serum T levels were not affected by pinealectomy.