Several cDNA clones for the human type II procollagen mRNA were isolated from a cartilage cDNA library. Six of the clones containing the longest inserts were subjected to restriction site mapping for alignment. All these six clones extended to the poly A tail. The longest clone, containing a 1470 bp insert, was named pHCAR3. Sequencing of pHCAR3 made it clear that neither of the two canonical AATAAA sequences of the human type II collagen gene is used as the polyadenylation signal. Two 60 bp stretches of high interspecies homology terminating in a hexanucleotide ATTAAA, located 23 nucleotides upstream of the poly A tail, apparently have an important role in determining the single polyadenylation signal for this gene. S1 protection experiments confirmed these observations.