Gonadotropin subunit mRNA levels rise after castration, coincident with a period of increased GnRH input to the pituitary. In addition to increased levels of gonadotropin mRNAs, we observed that the sizes of the alpha and LH beta mRNAs were increased after ovariectomy (OVX) of rats. To determine whether these changes occurred in the 5' (alternate transcriptional start site or splicing)- or 3' (altered polyadenylation)-end of the molecules, mRNAs were cleaved using oligonucleotide-directed RNase-H digestion, and the fragments were analyzed by Northern blot, using probes specific to the 5'- and 3'-segments of each transcript. After OVX, there was no change in the sizes of the 5'-segments of LH beta, FSH beta, and alpha-subunit mRNAs. However, the LH beta and alpha-subunit 3'-fragments were increased in size, indicating a shift to more adenylated forms of the LH beta and alpha transcripts. For FSH beta, the 3'-fragment bands were more diffuse than for LH beta or alpha-subunit, and no alteration in the lengths of FSH beta poly(A) tails were detected. A perifused pituitary cell system was used to determine whether pulses of GnRH were sufficient to cause modifications of polyadenylation. GnRH was administered as hourly 10-nM pulses for 4-12 h. Time-dependent increases in the sizes of LH beta and alpha-subunit mRNAs were observed in GnRH-treated cells compared to cells receiving no GnRH. Changes in the lengths of LH beta and alpha-subunit mRNAs were shown to be due to increased polyadenylation, and there was no observable change in polyadenylation of FSH beta mRNA. In addition, no changes were observed in the size of the 3'-fragments of PRL or beta-actin mRNAs. These data demonstrate that pulsatile GnRH administered in vitro elicits specific increases in the lengths of the LH beta and alpha-subunit mRNA poly(A) tails. Similar changes occur after OVX. Thus, in addition to transcriptional stimulation of the gonadotropin gene, GnRH modifies gonadotropin mRNAs at a posttranscriptional level.