The mechanisms controlling the pulsatile release of gonadotropins in prepubertal heifers are not completely understood. We examined the role of excitatory amino acid neurotransmitters, via activation of N-methyl-D-aspartate (NMDA) receptors, in the control of pulsatile LH and FSH release during prepubertal development in heifers. Hereford heifer calves received 4.7 mg/kg of N-methyl-D,L-aspartic acid (NMA), a potent NMDA receptor agonist (n = 5, i.v.), or saline (n = 5, i.v.), as single doses, at 4, 8, 12, 24, 36, and 48 wk of age. Blood samples were collected every 15 min, for 1 h before and 9 h after injection, on the days of treatment. Injection of NMA resulted in an acute release of LH (p < 0.001) in 0, 3, 3, 4, 5, and 5 calves (p < 0.01) and of FSH (p < 0.001) in 0, 1, 2, 4, 3, and 2 calves at 4, 8, 12, 24, 36, and 48 wk of age, respectively. The peak response of LH and FSH release to NMA was at 15 min posttreatment, and these peak responses were highest at 36 wk of age (p < 0.05). We suggest that neuroexcitatory amino acids, through NMDA receptors, are involved in prepubertal development of LH and FSH secretion in heifer calves.