The purpose of this study was to examine the 24-hour variation in endogenous tyrosine hydroxylase (TH) activity and thus catecholamine (CA) synthesis in the hamster and rat pineal gland. To determine CA synthesis a time course of the accumulation of dihydroxyphenylalanine (DOPA) after DOPA decarboxylase inhibition with m-hydroxybenzylhydrazine (NSD-1015) was measured at 0, 15 and 30 min by liquid chromatography with electrochemical detection. Animals were under long photoperiods (LD 14:10; lights on at 06.00 h). In the hamster pineal gland, CA synthesis was greater in the dark (24.00 h) (0.017 ng/pineal/min) than in the light (12.00 h) (0.008 ng/pineal/min). Similarly, CA synthesis in the rat pineal was 0.037 ng/pineal/min (dark) and 0.005 ng/pineal/min (light). In a 24-hour study, animals were injected with NSD-1015,30 min prior to killing to determine if 24-hour changes were present in CA synthesis. In the hamster, DOPA in the dark (p < 0.001) was significantly greater than in the light (1.33 ± 0.42 ng/pineal at 06.00 h; 0.33 ± 0.07 at 13.00 h) in this study. No significant difference was measured in norepinephrine (NE) concentration during this 24-hour period. In the rat, DOPA accumulation was significantly different (p < 0.001) in the dark as compared to the light (0.86 ± 0.09 ng/pineal at 03.00 h; 0.21 ± 0.08 at 12.30 h). Within this 24-hour period, NE concentration fluctuated significantly between 2.28 ± 0.33 ng/pineal (15.30 h) to 4.65 ± 0.59 (05.30 h). These results indicate for the first time a definite 24-hour rhythm in endogenous TH activity and NE synthesis in the hamster pineal gland even though NE content does not change. In addition, a 24-hour change in CA synthesis and content is present in the rat pineal gland.