The expression of the human immunodeficiency virus type 1 mRNAs containing the Rev-responsive element is regulated at the posttranscriptional level by the viral Rev protein. Rev increases the nucleocytoplasmic export of these mRNAs, leading to high expression. Using in situ hybridization and electron microscopy, we investigated the localization of a subgenomic gag mRNA in the absence and presence of Rev. In addition to the previously shown cytoplasmic accumulation of the Rev-dependent mRNA, we observed that in the presence of Rev the nuclear gag mRNA accumulates nonrandomly and forms specific localization patterns at the nuclear membrane and in the nucleoplasm. Cellular mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase were not found to form such patterns. These data suggest that Rev leads the gag mRNA to specific subnuclear locations, which further supports the transport function of Rev.