On the day of proestrus in the rat, rising plasma levels of estradiol (E) act in concert with progesterone (P) to trigger a preovulatory release of gonadotropins. Cellular levels of galanin mRNA in GnRH neurons are increased in association with the proestrous surge of gonadotropin secretion; however, the relative contribution made by E and P to the induction of galanin mRNA expression in GnRH neurons is unknown. We investigated the role of E and P in the induction of galanin gene expression in GnRH neurons by examining the effects of different combinations of E (estradiol benzoate; 50 μg and P (5 mg)) on the LH surge and the concomitant induction of galanin mRNA in GnRH neurons. We sacrificed ovariectomized adult rats after 1 of 4 treatments: Group 1: vehicle control (n=6); Group 2: P alone (n=7) Group 3: E alone (n=7); Group 4: combined E/P (n=6); the animals were killed at 18.00 h at the time of the LH surge. The brains from these animals were processed by double-label in situ hybridization to allow measurement of galanin mRNA levels in GnRH neurons. GnRH neurons were identified with a digoxigenin-labeled cRNA probe for GnRH mRNA, and galanin mRNA was detected and measured simultaneously with an 35S-labeled cRNA probe coupled with computerized grain counting. Estimation of cellular levels of GnRH mRNA was accomplished with single-label in situ hybridization, an 35S-labeled GnRH cRNA probe and computerized grain counting. We observed a 3-fold induction of galanin mRNA in the GnRH neurons of animals treated with E alone compared with those treated with the vehicle alone (vehicle: 13±2 vs E: 42±4 grains/cell (g/c); P<0.01); LH levels in the E-treated animals were elevated, albeit moderately, with respect to the vehicle controls. Compared with vehicle-treated animals, those treated with the combination of E and P showed a 5-fold induction of galanin mRNA in GnRH neurons (68±9 g/c), which was significantly (P<0.01) greater than that observed in the animals treated with E alone; in addition, the magnitude of the LH surge was much greater (P<0.05) in the E/P-treated group compared with the E alone group. In contrast, compared to the vehicle controls, animals treated with P alone (15±2 g/c) showed no discernable effect on galanin mRNA levels; moreover, no LH surge occurred in the P alone group. Neither the number of identified GnRH cells nor their content of GnRH mRNA differed significantly among the experimental groups (GnRH mRNA signal: vehicle controls: 153±6 vs E: 159±6 vs E/P: 153±3 vs P: 148±8 g/c). We conclude that while E is the primary ovarian signal inducing galanin mRNA expression in GnRH neurons and the LH surge itself, P plays a facilitatory role in both of these processes.