To further evaluate nutrient regulation of GRF synthesis, we measured hypothalamic preproGRF messenger (m) RNA in food-deprived rats refed diets varying in nutrient composition by nuclease protection analysis. Adult male Sprague-Dawley rats were allowed free access to food (Fed), food deprived for 72 h (72-h FD), or 72 h FD then refed for 72 h with either a normal (NF) diet or isocaloric diets containing no protein (PF), carbohydrate (CF), or fat (FF). Seventy-two-hour FD rats displayed the expected 80% reduction in hypothalamic preproGRF mRNA. Upon refeeding, levels were normalized in rats refed NF, CF, or FF diets. In contrast, preproGRF mRNA in rats refed a PF diet was similar to that in 72-h FD rats. Rats refed a PF diet failed to gain weight and consumed less food than animals refed NF, CF, or FF diets. However, the lack of the GRF response to the PF diet was due to protein deprivation rather than caloric restriction, since hypothalamic preproGRF mRNA returned to 66% of Fed values in rats refed an equivalent amount (grams per day) of a NF diet. In 72-h FD rats refed isocaloric diets containing 4%, 8%, or 12% protein, preproGRF mRNA was restored to Fed values in a protein concentration-dependent manner being completely restored by the 12% diet. A lack of dietary protein was sufficient to regulate hypothalamic preproGRF mRNA since feeding rats a PF diet without prior food deprivation resulted in 70% reduction in preproGRF mRNA, whereas CF and FF diets were without effect. These data indicate that decreased hypothalamic preproGRF mRNA expression in 72-h FD rats occurs as a result of dietary protein deprivation.