We have characterized an inhibitory RNA element in the human immunodeficiency virus type 1 (HIV-1) gag coding sequence that prevents gag expression. The inhibition exerted by this element could be overcome by the presence of the Rev-responsive element in cis and of Rev protein in trans. To understand the mechanism of function, we inactivated the inhibitory element by mutagenesis while maintaining an intact gag coding region. A constitutive high level of Rev-independent gag expression was achieved only after the introduction of 28 point mutations over a large region of 270 nucleotides within the gag coding region. To our knowledge, this is the first demonstration of inactivation of a negative RNA element within a coding region without alteration of the expressed protein. Elimination of the inhibitory element in the p17gag region, named INS-1, offered the opportunity to detect a second inhibitory element in the gag-pol region. The presence of either INS element is sufficient to inhibit gag expression, demonstrating that multiple INS elements acting independently can inhibit HIV RNA expression. Expression of gag from Rous sarcoma virus, a retrovirus that does not require Rev-like regulatory proteins, revealed that the Rous sarcoma virus p19gag region does not contain inhibitory elements. These results demonstrate the presence of a strong inhibitory element acting at the level of mRNA and provide a general method for the removal of such elements from mRNA coding regions. The inhibitory element functions in the absence of any HIV-1 proteins, suggesting that cellular factors are responsible for this inhibition.