Male Syrian hamsters were kept under either 14 h light/10 h dark (lights on at 06.30 h) or 2 h light/22 h dark (lights on at 14.30 h) photoperiods. Groups of hamsters under each photoperiod were rendered hypothyroid by addition of 0.4% thiourea to the drinking water. These hamsters received, in addition, either a daily evening injection of saline or a daily injection of 25 µg melatonin in saline. Groups of intact controls and pinealectomized control hamsters were also maintained under the two photoperiodic conditions. After 10 weeks under the different conditions the hamsters were killed by decapitation, and serum samples assayed for thyroxin, thyroid-stimulating hormone (TSH), and prolactin (PRL). Pituitary extracts were assayed for TSH and PRL. Hypothyroidism in hamsters receiving thiourea was confirmed by radioimmunoassay data showing low serum thyroxin and greatly elevated serum TSH concentrations. Melatonin injections resulted in significant depression of serum TSH in thiourea-treated hamsters under short photoperiod compared to saline-injected controls. Both melatonin injections and short photoperiod resulted in a significant reduction of pituitary TSH in hamsters on thiourea compared to values obtained from similarly treated animals under the 14 h light/10 h dark photoperiod. Hypothalamic concentrations of thyrotropin-releasing hormone (TRH) were significantly elevated by melatonin injections and by short photoperiodic conditions, but not by thiourea administration. The short photoperiod resulted in testicular involution which was completely reversed by pinealectomy and partially reversed (to 53% of controls) by thiourea treatment. Involution of gonads was complete in thiourea-treated animals under short photoperiod, if they received melatonin injections. It is concluded that the inhibitory effect of melatonin on the pituitary-thyroid axis is the result of an inhibition of release of hypothalamic TRH. Since incomplete gonadal involution was observed in hypothyroid hamsters under short photoperiod, the data suggest a thyroxin requirement for complete gonadal involution. The hypothesis that inhibition of both TSH and PRL secretion is the result of decreased release of hypothalamic TRH is discussed