The retroviral RNA genome is targeted for incorporation into the nascent virion particle by the psi region, a specific block of RNA sequences near the 5' end. A number of deletions and linker insertion mutations were introduced into the psi region of cloned DNA of the Moloney murine leukemia virus, and the mutants were introduced into cells in culture and tested for their ability to direct the assembly of virions and the packaging of viral RNA. Only a small portion of the psi region was important for packaging, containing the so-called stem-loops C and D. Additional mutants were used to demonstrate that the base pairing of stem D, and the sequence of loop D, were essential for normal packaging of the RNA. Two mutants with alterations near the 5' splice donor were also replication-defective, probably due to effects on gene expression. The results allow a high-resolution definition of the RNA structures required during virus replication in culture.