Control of HIV-1 RNA processing and transport are critical to the successful replication of the virus. In previous work, we identified a region within the HIV-1 env that is involved in mediating nuclear retention of unspliced viral RNA. To define this sequence further and identify elements required for function, deletion mutagenesis was carried out. Progressive 5' and 3' deletions map the nuclear retention sequence (NRS) within the intron between nts 8281 and 8381. While deletion of sequences comprising the 3'ss had no effect, removal of the 5'ss resulted in cytoplasmic accumulation of unspliced RNA. Sequence analysis determined that the region corresponding to the NRS is highly conserved among HIV-1 strains. To evaluate whether this NRS interacts with cellular factors, RNA electrophoretic mobility shift assays (REMSA) were performed. We show that the NRS specifically interacts with cellular factors present in HeLa nuclear extracts, and, by UV crosslinking, correlates with the binding of a 49-kDa protein. Immunoprecipitation of the UV crosslinked products determined that this 49-kDa protein corresponds to hnRNP C.