Nuclei of cells infected with Moloney murine leukemia virus (MoMuLV) were examined for the presence of gag proteins. This analysis was performed in conjunction with other studies suggesting a possible role for gag proteins in regulating nuclear events relating to processing and/or transport of viral genomic RNA. We detected Pr65gag and a p30-related protein in a nuclear fraction of infected cells. We also found evidence that a highly conserved amino acid sequence, which is shared by p30 and U1 small nuclear ribonucleoprotein 70-kDa protein, is a component of the nuclear targeting sequence for Pr65gag. Immunoelectron microscopy studies with a monoclonal anti-p12 antibody established that approximately 18% of gag-containing proteins of MoMuLV are located in the nucleus. Such gag-containing proteins from a mutant MoMuLV that lacks N-terminal myristic acid had greater affinity for the nucleus, suggesting that fatty acid acylation of Pr65gag plays a role in overcoming the proposed nuclear transport signal. The possible roles that nuclear gag proteins may play in retroviral replication are discussed.