Partial Root Zone Drying (PRD) is an irrigation strategy which involves the alternate drying and wetting of sub-sections of the plant root zone. Savings in crop water use productivity from PRD is a result of changes in the plant biochemical and physiological response from the imposed soil moisture gradient. However, an understanding of the relationship between the soil moisture gradient and plant response is required before PRD can be used to improve crop water use productivity. The objective of this study was to investigate the response of cotton (Gossypium hirsutum L.) to a soil moisture gradient imposed across the root zone. Cotton plants were grown under greenhouse conditions in split pots. Control treatment pots were well watered on both sides of the split pots and a Non-alternated PRD treatment had water applied to only one side of the split pot with the other allowed to dry over a 24 day period. Soil moisture potential on each side of the split pots was measured along with changes in plant stem sap abscisic acid, sap pH, and stomatal conductance. The imposed soil moisture gradient resulted in a four-fold increase in xylem sap abscisic acid concentration, peaking at a soil moisture potential of -2360 kPa. However, this soil moisture gradient did not produce any significant (P<0.05) difference in either xylem sap pH or stomatal conductance. These findings suggest that it may not be possible to maintain the plant water status of cotton grown under commercial field conditions and simultaneously impose a sufficiently large soil moisture gradient across the root zone to induce a PRD response.