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The coordination between phytohormones regulation, stomatal behaviour (stomatal index and opening/closing) and gas exchange are potent determinants of plant survival under drought stress. However, we found a knowledge gap in the mechanism regulating the fine-tuning of these features during drought. In order to address this we evaluated gas exchange, stomatal behaviour and endogenous phytohormones content in two cotton varieties (LRA-5166 and NBRI-67) differing in drought sensitivity during water deficit conditions. Variety specific differences were recorded in net photosynthesis rate (A), transpiration rate (E) and stomatal conductance (gs) with significantly less decrease in drought tolerant LRA-5166 than drought sensitive NBRI-67. The abscisic acid (ABA) accumulation was significantly increased in LRA-5166 while reduced in NBRI-67 under water deficit, which was accompanied by relatively less reduced 6-benzylaminopurine (6-BAP) level in LRA-5166 than NBRI-67. Thus, improved ABA/6-BAP ratio was observed in both the varieties of cotton. Critically, LRA-5166 has reduced stomatal index, aperture size and significantly higher A and intrinsic water use efficiency (WUEi), thus higher drought tolerance than NBRI-67. Furthermore, we found that endogenous ABA predominantly maintains the stomatal behaviour and regulates its physiology either by antagonizing 6-BAP or alone to coordinate with water deficit signals. Overall, our findings describe a new insight as to how drought modulates endogenous ABA and 6-BAP homeostasis in cotton leaf and the mechanism of stomatal regulation by ABA and 6-BAP in cotton. [ABSTRACT FROM AUTHOR]