This study examined the effects of two novel melatonin analogues in adult Djungarian hamsters housed under long photoperiod (LD 16:8). Daily injection (10 μg, s.c.) of either melatonin, 5-methoxy N-butanoyltryptamine (bMT), or 5-methyl N-butanoyltryptamine (5-MebT) 3 hr before lights off for 8 weeks led to a significant decrease in paired testis weight compared to vehicle-injected controls. The reduction in testis weight was of similar magnitude with melatonin and bMT, but 5-MebT was not as effective. The affinity of the analogues was determined in competition experiments using chicken brain membranes and 2-[125I]iodomelatonin (2-[125I]aMT). Replacing the N-acetyl side-chain of melatonin with an N-butanoyl group increased affinity for the chicken brain 2-[125I]aMT binding site, but exchanging the 5-methoxy group of melatonin for a 5-methyl group reduced affinity. These studies show that these analogues not only inhibit 2-[125I]aMT binding in vitro but also mimic melatonin's antigonadal activity in vivo.