Alternative RNA processing of the human fibroblast growth factor receptor-1 transcript results in receptor forms that vary in their affinity for fibroblast growth factor. An alternative RNA processing event involving recognition of the alpha-exon is deregulated during neoplastic transformation of glial cells. We have previously established a splicing reporter/transfection cell culture model system to identify sequences involved in recognition of this exon. In this study, the system was used to identify two sequence elements that differentially function to regulate splicing of this exon. Exclusion of the alpha-exon in glioblastoma cells specifically required the downstream intron sequence comprising the 5'-splice site. Replacement or mutation of this sequence increasing complementarity to U1 RNA resulted in enhanced exon recognition in SNB-19 glioblastoma cells. Sequences within the exon were found to be required for alpha-exon inclusion. Deletion and gain-of-function experiments identified a 69-nucleotide exon sequence that was specifically required for alpha-exon inclusion. These studies indicate that multiple sequences are required for the regulated recognition of the alpha-exon.