The tenascin-R (TN-R) gene encodes a multidomain extracellular matrix protein belonging to the tenascin family, previously detected only in the central nervous system, In this report, we describe the structure of the 5' region of the human TN-R gene and characterize the activity of its promoter, We cloned two previously unreported nontranslated exons (exons 1 and 2, 539 and 101 bp in length, respectively) separated by a large (greater than or equal to 40-kb) intron, The intron between exons 2 and 3 (containing the ATG codon) is 122 kb in length, Tenascin-R transcripts in fetal, adult, and neoplastic human brain contain both exons 1 and 2, as demonstrated by S1 nuclease analysis and reverse transcriptase-polymerase chain reaction, The human TN-R promoter displays relatively unusual features in terms of sequence in that it lacks any TATA box, CAAT box, GC-rich regions, or initiator element, The promoter displays its activity only in cultured cells of neural and glial origin, not in transformed epithelial cells and melanoma cells, All the elements required for the full and cell-specific activity of the promoter are contained in the 57-bp sequence closest to the transcription startpoint.