Previously, we demonstrated that excision of the GnRH first intron (intron A) was largely attenuated in non-GnRH-producing tissues but accelerated in GnRH neurons. In the present study, we examined the splicing rate of GnRH pre-mRNA in developing normal mice and adult hypogonadal mice. The preoptic area and cerebral cortex were removed from mice at ages 1-7 wk. GnRH pre-mRNA splicing was examined by competitive RT-PCR using a variety of primer sets. The ratio of mature mRNA to intron A-containing RNA species in the preoptic area was lowest in 1- and 2-wk-old mice, significantly augmented in 3-wk-old mice, and further increased until adulthood. In contrast, the ratio of mRNA to intron A-containing RNA in the cerebral cortex was extremely low, drastically decreased in 3-wk-old mice, and remained at low levels until adulthood. These data indicate a preoptic area-specific increase in intron A excision during development. Intron B or C excision in the preoptic area was not significantly changed during development. To elucidate the possible involvement of the exonic splicing enhancers located in GnRH exons 3 and 4 in the developmental increase in intron A excision, we examined the splicing rate of GnRH pre-mRNA in hypogonadal mice whose GnRH exons 3 and 4 were truncated. The intron A excision in the preoptic area of hypogonadal mice was significantly lower than that of normal mice but similar to that in other tissues, such as cerebral cortex and olfactory bulb. To support the functional relevance of intron A-containing RNA species, we examined the translation efficiency of intron A-containing RNA. Insertion of intron A sequence into the upstream portion of the luciferase open reading frame significantly decreased translation efficiency. The present study demonstrates that intron A excision in the preoptic area is developmentally regulated in normal mice but largely attenuated in hypogonadal mice, indicating the functional importance of intron A excision in GnRH pre-mRNA splicing.