Different soybean cultivars (Williams 82, Union, Jindou 21, Long Huang 1, Long Huang 2) were exposed to drying soil, to investigate whether endogenous abscisic acid (ABA) concentrations and leaf water relations regulated stomatal behaviour. We measured ABA concentrations in xylem and tissue of the first and second trifoliate leaves respectively; stomatal conductance (gs) and leaf water potential (Ψleaf) in both leaves; and water content in soil. Cultivar variation in leaf area and gs caused different rates of soil drying, but gs and Ψleaf declined similarly with soil drying in all cultivars. Variation in leaf xylem ABA concentration better explained stomatal responses than foliar ABA concentration in some cultivars, and was highly correlated with stomatal conductance. Xylem ABA concentration in well-watered soil was highest in Union, and in drying soil was lowest in Jindou 21 and Long Huang 2, although the latter had the highest foliar ABA concentrations. Jindou 21 accumulated lower xylem ABA concentrations than other cultivars as soil moisture or Ψleaf decreased, but its stomatal sensitivity to xylem ABA was greater. Because cultivars varied in both ABA accumulation and stomatal sensitivity to ABA, but had similar stomatal sensitivity to Ψleaf, leaf water relations seem more important in regulating stomatal closure of soybean.