The RNA synthesis of a single-stranded, multisegmented viral genome has been investigated in nonpermissive host cells. In HeLa cells, both single- and double-stranded influenza viral RNA were made and accumulated, and synthesis was completed by 15 hours. The evidence indicated that all pieces of the genome were synthesized. At least as much viral RNA was synthesized in HeLa cells as in permissive chick embryo fibroblasts cells. Explanations for this nonpermissive infection based on reduced synthesis of viral RNA or rapid destruction of newly synthesized RNA, uncontrolled synthesis of RNA, or partial synthesis of the genome appear to have been ruled out by this investigation. The persistently high amounts of double-stranded RNA, combined with the reported lack of dispersion of nucleocapsid protein from the nuclear region of the HeLa cell are consistent with a failure of assembly of the virion in this host. This nonpermissive infection of HeLa cells with influenza virus may offer a unique system for the accumulation of the intermediates involved in the assembly of a complex RNA viral genome.