We have isolated several overlapping genomic clones which contain the 5' terminal portion of the rat pro-alpha 1 (II) chain gene. These clones span about 20 kilobases (kb) of contiguous DNA containing 15 kb of the gene and 5 kb of the 5' flanking sequence. Electron microscopic analysis of mRNA-DNA hybrids by R-looping shows that collectively these clones contain 16 exons which code for approximately one-third of the pro-alpha 1 (II) chain. The sizes of the exons are small, except for the first exon which is relatively large. The nucleotide sequence of the first exon and the 1000 base pairs (bp) preceding it was determined. The first exon contains a 150-bp untranslated segment and an 85-bp sequence coding for the signal peptide and a part of the NH2-terminal propeptide of type II collagen. The segment preceding the transcription initiation site contains the “TATA” box and several G + C-rich stretches, whereas the “CAT” box is not evident between -70 and -120. The hexanucleotide sequence 5'-GGGCGG-3' is found in three different places between -200 and the TATA box. The inverted complement sequence of this hexanucleotide, 5'-CCGCCC-3', is located around both -220 and -450. The hexanucleotide and its inverted sequence have been found previously in the promoter region of the tk gene of herpes virus. These sequences are known to function in a mutually dependent manner as transcription signals for the tk gene; thus, they may play a role in determining the level of transcription of this cartilage gene. The hexanucleotide, 5'-CCGCCC-3', is also found in the 21-base pair repeats of the SV40 promoter and the promoter region of hydroxymethylglutaryl-CoA reductase gene. The sequence 5'-GTGGTTAGA-3' located around -280 is identical to the “core” sequence that has been reported as enhancer element in both viral and cellular genes. These unusual structures may be related to the tissue-specific expression of this gene.