Galanin is colocalized with GnRH, and its expression in these neurons is enhanced at proestrus, a time of activation of GnRH neurons. We tested the hypothesis that the expression of both the GnRH and galanin mRNAs in GnRH neurons decrease during lactation in the rat, a reproductive state characterized by reduced gonadotropin secretion. For double label in situ hybridization, GnRH mRNA was detected with an antisense cRNA probe labeled with the hapten digoxigenin, whereas galanin mRNA was detected with a cRNA probe labeled with 35S. The number of silver grains deposited over a digoxigenin-labeled cell body provided an index of galanin mRNA levels in GnRH cells. We observed a 60% reduction in signal (grains per cell) for galanin mRNA in GnRH neurons of lactating animals compared with those of diestrus animals (P < 0.004), with no difference in the number of GnRH neurons between groups. To compare cellular GnRH mRNA content between groups, we used single label in situ hybridization and image analysis. Signal levels (grains per cell) for GnRH mRNA were not different between diestrus and lactating animals in either an initial (diestrus, 121.4 +/- 5.9; lactation, 117.3 +/- 8.0; P > 0.7) or in a subsequent trial (diestrus, 184.0 +/- 10.4; lactation, 197.5 +/- 13.0; P > 0.7). To confirm and extend these findings, we used a RNAse protection assay to measure and compare the content of GnRH mRNA in hypothalamic fragments between diestrus and lactating animals. The concentration of GnRH mRNA (picograms of mRNA per 25 micrograms total RNA) was not different between the two groups (diestrus, 1.21 +/- 0.25; lactation, 1.25 +/- 0.13; P > 0.7). A determination of the total GnRH peptide content by RIA in a separate set of hypothalamic dissections revealed no difference between groups in the level of GnRH content (nanograms) per hypothalamus (diestrus, 6.0 +/- 0.6; lactation, 5.7 +/- 0.4; P > 0.4). We conclude that galanin mRNA expression in GnRH neurons of the rat is diminished during lactation, whereas GnRH expression continues unabated. This decrease in galanin gene expression associated with lactation may lead to decreased synthesis and secretion of galanin, which, in turn, could diminish the pulsatile secretion of GnRH or reduce its activity at the pituitary.