Fusaric acid (FA) is produced by several Fusarium species that commonly infect cereal grains and other agricultural commodities. FA in the feed of nursing dams is lactationally transferred to the suckling offspring and alters serotonin (5-hydroxytryptamine, 5HT) in the pineal gland of the neonate rat. 5HT is involved in melatonin (MEL) production by the pineal gland. MEL is a hormone important in reproduction and seasonality in animals. Therefore, the effects of FA on MEL in the serum and pineal gland of male and female 21-d-old weanling rats from dams on an FA diet were studied. MEL was measured by enzyme-linked immunosorbent assay (ELISA), which was standardized for directly measuring MEL in rat serum and pineal homogenates. At 200 ppm in the diet of nursing dams, FA increased serum MEL in both sexes. Results obtained from ELISA were supported by high-performance liquid chromatography (HPLC) analysis with fluorescence detection. MEL analysis of the pineal gland homogenates by ELISA and HPLC supported observations in the serum. Analogously, in pineal cell monolayer cultures, FA at 1 M and 100 M concentrations increased MEL in a dose-dependent manner as compared to the control cells. This is the first report that FA increases MEL in vivo and in vitro and suggests that FA contamination of diets may affect mechanisms involving MEL synthesis.