"Splicing" of the precursor to an adenovirus mRNA was accomplished in isolated cell-free extracts. Nuclei were prepared from hypotonically swollen cells that had been labeled with [3H]uridine for 10 min prior to nuclear isolation. Addition of a "cytoplasmic" fraction was required for the splicing to occur. The nuclear precursor, a poly(A)-terminated RNA molecule approximately 5 kilobases long, contained sequences complementary to the 58.5--75.9 region of the adenovirus 2 genome, including those sequences spliced out of the mature mRNA molecule. The in vitro spliced product was a poly(A)-terminated RNA molecule identical in size to the cytoplasmic 72,000 Mr protein mRNA (2 kilobases long) in which the sequences encoded in the 70.7--75.9 region of the viral genome were spliced to those encoded at 58.7--65.6, with the sequences encoded at 66.1--70.7 deleted.