The glutamate receptor gene GluR-4 is proposed to generate two spliced isoforms (Sommer et al., 1990). Screening a rat cerebellar cDNA library, we have now identified a third type of transcript derived from GluR-4 gene by differential RNA processing. This transcript encodes a protein with a “flop” module between transmembrane regions 3 and 4, but with a C-terminus segment of 36 amino acids different from the previously described GluR-4 flip/flop cDNAs. This subunit was therefore designated as GluR-4c flop. Transcripts synthesized in vitro from GluR- 4c cDNA form kainate/AMPA-activated channels when expressed in Xenopus oocytes. The current-voltage relationship for kainate-evoked responses in oocytes injected with GluR-4c showed strong inward rectification. The different transcripts derived from the GluR-4 gene were studied on Northern blots hybridized with either a cDNA probe or oligonucleotides specific for the GluR-4 flip/flop and C-terminal domains. Three transcripts of 6.2, 4.2, and 3.0 kilobases (kb) derived from the GluR-4 gene were identified on Northern blots containing total RNA prepared from different brain regions, using a cDNA probe or an oligonucleotide corresponding to the N-terminal region common to all transcripts. These transcripts were much more abundant in the cerebellum than in other brain areas, and their levels increased during cerebellar development. The maximal increase was observed between postnatal days 1 and 20, an age corresponding to the division and maturation of granule neurons. The flip/flop and the C-terminal oligonucleotides hybridized to the two higher molecular weight transcripts but did not hybridize to the small RNA. Interestingly, using cerebellar cells that were cultured for up to 12 d, we observed that the three transcripts are present in granule neurons, but that astrocytes only express the 6.2 and the 4.2 kb transcripts. The 3.0 kb transcript accumulates in cerebellar granule cells during development in vitro. Furthermore, in situ hybridization histochemistry revealed that the GluR-4c transcripts are preferentially expressed in cerebellar granule cells and Bergmann glial cells, whereas the expression of GluR-4 flip mRNAs is restricted to Bergmann glial cells. Interestingly, we also show that granule cells already express GluR-4c in the premigratory zone of the external granular layer, indicating that intrinsic or highly localized cues induce GluR-4c expression before these cells reach their final position.