We have previously studied sequence heterogeneity of HIV-1 Rev response element (RRE), and showed uneven variations in different stem-loops of both primary sequence and secondary structure. Here we studied the functional variation of RRE clones from a set of 10 primary isolates, and demonstrated a variation in the function of these RRE clones on the expression of Gag proteins from a truncated HIV-1 genome. The difference in Gag level was, in part, if not exclusively, resulted from the differential efficiency of RNA transport and enhancing of translation. These data suggested that variation of HIV-1 RRE may play a role in regulation of viral replication rate in HIV-1 primary isolates.