In the polyoma virus (Py) transformed 3B rat cell line the Py oncogene and adjacent cellular DNA are amplified in arrays of very large inverted duplications. A region of the 3B amplified DNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome (YAC) vector, designated y3B01. Analysis of the y3B01 cloned insert revealed it contained a large inverted duplicated DNA region which was approximately 400 kb in size (two palindromic arms of about 200 kb). At least 420 kb of the 550 kb YAC insert has been identified as being derived from the 3B amplified DNA and the amplicon in 3B cells is at least 220 kb in size. No DNA instability of the y3B01 YAC clone was detected. The y3B01 DNA replicated as efficiently as yeast chromosomes and was structurally stable in yeast cells during more than 30 cell divisions. Comparison of the restriction endonuclease maps of the inverted duplicated region of the y3B01 DNA insert and the amplified 3B genomic DNA did not reveal any gross differences suggesting that no rearrangements had occurred during or after the cloning into the YAC vector. These results suggest that large inverted duplications, which can show instability in prokaryotic cloning systems, can be stably cloned and maintained in YAC vectors in yeast.