Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.