Among the regulators of human immunodeficiency virus (HIV) replication is the cellular transcription factor NF-kappaB, whose activity is regulated through inhibition by IkappaB family members. We have shown previously that I kappaB-alpha inhibits HIV type 1 (HIV-1) replication, and unexpectedly, IkappaB-alpha was found both to suppress HIV-1 transcription and to inhibit Rev function. The relative contributions and specificities of these mechanisms to HIV replication were unknown. Here, we report that the region of IkappaB-alpha which blocks Rev function is separable from that required for inhibition of NF-kappaB. Molecular mutagenesis revealed that the N terminus of IkappaB-alpha is required for inhibition of Rev function, whereas mutants lacking the N terminus retained the ability to inhibit NF-kappaB function. Interestingly, the nuclear export sequence of IkappaB-alpha was not required for inhibition of Rev or NF-kappaB function in mammalian transfection assays. Conversely, the C terminus of IkappaB-alpha was not required for the inhibition of Rev, while deletion of this region resulted in a loss of NF-kappaB inhibition. Another IkappaB family member with a distinct amino-terminal sequence, IkappaB-beta, inhibited NF-kappaB but not Rev function. These studies indicate that the inhibition of Rev by IkappaB-alpha is independent of NF-kappaB. Mutants defective in inhibition of either Rev or NF-kappaB retained the ability to inhibit HIV-1 replication, suggesting that both functions may contribute to the inhibition of HIV replication by I kappaB-alpha.