The precursor of gonadotropin-releasing hormone (GnRH) and the 56-amino acid GnRH-associated peptide is encoded in an mRNA of about 560 bases in length. This mRNA derives from an approximately 4300-base pair-long gene consisting of four relatively short exons (denoted 1, 2, 3, and 4) and three large introns (A, B, and C). In this study, we characterized the order by which the three introns are spliced from the primary transcript and processing intermediates to give rise to a mature mRNA and evaluated the potential role of gene transcription and pre-mRNA processing in the control of proGnRH mRNA levels in vivo. Nuclear and cytoplasmic RNA fractions isolated from rat preoptic area-anterior hypothalamus (POA-AH) and basal olfactory area (located rostral to the POA) were analyzed by 1) solution hybridization-RNase protection mapping using several RNA probes directed at various regions of the proGnRH gene and 2) reverse transcription-polymerase chain reaction using several oligonucleotide primers. Both types of analysis showed that proGnRH pre-mRNA processing begins with the splicing of intron B from the primary gene transcript. Hence, intron B is the ideal target for studying proGnRH primary transcript by in situ hybridization. Subsequent splicing of introns A and C appeared to take place in two alternative, although not equally prevalent pathways. Quantitative analysis indicated that the proGnRH hnRNA species constituted, on a mole basis, about 20% of the total gene transcripts in the POA-AH. The primary transcript alone constituted about 10% of the total gene transcripts in the POA-AH and as much as 20% in the basal olfactory area. The prospect of blockade of proGnRH hnRNA processing by means of hybridization with endogenous antisense RNAs (transcribed from the SH gene on the opposite strand of the same DNA locus) did not prove to be likely, as the SH transcripts were present at very low levels compared to any of the proGnRH RNA species. We conclude that the relatively large pool of proGnRH hnRNA may reflect a high rate of gene transcription and/or slow RNA processing.