The interactions of catecholamines with alpha 1B-adrenergic receptors (alpha 1B-AR) located on the surface of many cell types are responsible for physiologic and pathologic functions in mammalian systems. Transcription of the alpha 1B-AR gene leads to the expression of multiple alpha 1B-AR mRNAs that are distributed in a tissue-specific fashion. The purpose of this study was to define the 5'-untranslated regions of the multiple alpha 1B-AR gene transcripts. Evidence for a previously unidentified intron in the alpha 1B-AR gene upstream of the receptor open reading frame was obtained via rapid amplification of cDNA ends. A product was amplified and was found to be missing the nucleotide interval from -708 to -194, (+1 is the start of translation). Evidence for tissue-specific alternative intron splicing was obtained from ribonuclease protection assays and RT-PCR experiments. Using an RNA probe extending from -240 to +93 and including 45 nucleotides into the putative intron, a single protected fragment was detected in heart RNA while two protected fragments were detected in liver RNA. RT-PCR amplification of the region spanning the intron resulted in detection of two PCR products in liver RNA and no detectable product in heart RNA. These findings emphasize the complexity of alpha 1B-AR gene regulation and suggest that multiple alpha 1B-AR mRNAs with different 5'-UTRs may play a role in regulating alpha 1B-adrenergic responsiveness.