Protein 4.1B has been reported as a tumor suppressor in brain, but not in kidney, despite high expression in both tissues. Here we demonstrate that N-terminal variability in kidney and brain 4.1B isoforms arises through an unusual coupling of RNA processing events in the 5' region of the gene. We describe two transcriptional promoters at far upstream alternative exons 1A and 1B, and show that their respective transcripts splice differentially to exon 2'/2 in a manner that determines mRNA coding capacity. The consequence of this unique processing is that exon 1B transcripts initiate translation at AUG1 (in exon 2') and encode larger 4.1B isoforms with an N-terminal extension; exon 1A transcripts initiate translation at AUG2 (in exon 4) and encode smaller 4.1B isoforms. Tissue-specific differences in promoter utilization may thus explain the abundance of larger 4.1B isoforms in brain but not in kidney. In cell studies, differentiation of PC12 cells was accompanied by translocation of large protein 4.1B isoforms into the nucleus. We propose that first exon specification is coupled to downstream splicing events, generating 4.1B isoforms with diverse roles in kidney and brain physiology, and potentially unique functions in cell proliferation and tumor suppression.