AbstractOut of various subtypes of human immunodeficiency virus type 1 (HIV‐1), subtype B and C cause most of the infections worldwide. Clade specific differences have been reported in differences in clinical picture of HIV pathogenesis. Transcription of the HIV‐1 genome is regulated by the interaction of HIV Tat protein to the trans‐activation response (TAR) element. The differential binding of clade B and C Tat proteins to TAR and differences in activation of NF‐κB cascade leading to differential transactivation capacity and cytokine expression has been examined in this study. More stable Tat‐TAR complex formation by Tat‐C revealed by EMSA and higher TNF‐α expression shown by Tat‐C compared to Tat‐B leads to higher NF‐κB activation, which may be plausible cause for higher transactivation by Tat‐C as obtained by FACS analysis. This comparative study would be helpful in understanding the basic mechanism of clade specific Tat protein differences and their functional relationships. J. Med. Virol. 87:1334–1343, 2015. © 2015 Wiley Periodicals, Inc.