Degradation of amino acids by rumen microbes poses a challenge as the true benefit of amino acid supplementation is only exploited when the amino acid is presented to the small intestine for absorption. Our laboratory has previously demonstrated that prepartum intravenous (IV) infusions of 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin, increases circulating total calcium concentrations. This suggests potential for 5-HTP as a therapeutic management strategy for hypocalcemia prevention. However, whether 5-HTP can escape the rumen and be absorbed is unknown. The objective of this study was to determine if ruminal administration of 5-HTP increases circulating serotonin concentrations. The experiment was conducted as a 4 × 4 replicated Latin square using 4 nonlactating, nonpregnant, ruminally fistulated multiparous dairy cows. Experimental dosings of 5-HTP were administered on 2 consecutive days and given on a mg/kg of body weight basis. The resulting 4 treatments were saline infusion of 0 mg/kg 5-HTP (CON), IV infusion of 1 mg/kg 5-HTP (IV), 1 mg/kg intraruminal 5-HTP, and 2 mg/kg intraruminal 5-HTP. Whole blood was collected relative to administration of the second experimental dose for 3 continuous days with a 7-d washout period between treatment periods. Data were analyzed using the MIXED procedure of SAS with repeated measures. The ruminal 2 mg/kg 5-HTP treatment increased (P = 0.03) serotonin compared with the 0 mg/kg 5-HTP dose for 8 h after the second treatment. However, on d 3 and 4 of treatment, there were no differences between the CON (P > 0.05) and the 2 mg/kg 5-HTP administered intraruminally. The IV dose increased serotonin concentrations on d 2 compared with the 0 mg/kg 5-HTP dose, the ruminally dosed 1 mg/kg and 2 mg/kg 5-HTP treatments (P < 0.0001, P < 0.0001, and P = 0.0016, respectively). These data reveal that 2 mg/kg 5-HTP administered intraruminally acutely increased circulating serotonin concentrations. Future feeding experiments need to be done to determine the optimal 5-HTP dose for potential commercial use as a mitigation tool for hypocalcemia.