Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone with both direct and indirect functions in the maintenance of immune status under physiological and pathological conditions. In this study, 52 Holstein heifers were evaluated for the effects of a plasmid-mediated GHRH treatment on their immune function and on the morbidity and mortality of treated animals. In the third trimester of pregnancy, 32 heifers received 2.5 mg of a myogenic GHRH-expressing plasmid by intramuscular injection followed by electroporation, while 20 heifers were used as controls. No adverse effects were associated with either the plasmid delivery or GHRH expression. At 18 days after plasmid administration, GHRH-treated animals had increased numbers of CD2(+) alphabeta T-cells (P < 0.004), CD25(+)CD4(+) cells (P < 0.0007), and CD4(+)CD45R(+) cells (P < 0.016) compared to controls. These increases were maintained long term after treatment and correlated with plasmid expression. At 300 days post-GHRH therapy, CD45R(+)/CD45R0(-) naïve lymphocytes were significantly increased in frequency (P < 0.05). Natural killer lymphocytes (CD3(-)CD2(+)) were also increased. As a consequence of improved health status, body condition scores of treated animals improved (3.55 vs. 3.35, P < 0.0001). Hoof pathology was also reduced with treatment. The mortality of heifers was decreased (3% vs. 20% in controls, P < 0.003). Collectively, these results indicate that the myogenic GHRH plasmid can be successfully electrotransferred into a 500-kg mammal and expressed for prolonged periods of time, ensuring physiological levels of GHRH. The plasmid injection followed by electroporation could prove an efficient method for the systemic production of therapeutic proteins and may provide a useful means for basic research in relevant animal models.