The objective of this study was to test the hypothesis that root of maguey (Agave salmiana Otto ex Salm-Dyck) seedlings reacts during the first 24 h to low substrate water potential (PsiW), by anatomical modifications. Three-4 cm root length seedlings were planted in vermiculite for 24 h at PsiW between -0.03 and -2.35 MPa. Root dimensions, proline content and anatomy were evaluated. Substrate PsiW between -0.65 and -2.35 MPa did not significantly affect longitudinal root growth. However, proline content significantly increased from 1.6 to 2.1 micromoles mg(-1). Significant reductions of transverse root area (41%), thickness of mucilage covering the epidermis (47%), thickness of epidermis (between 15 and 46%), area of the parenchyma (between 35 and 41%) and number of vessels (up to 28%) were observed with PsiW of -2.35 MPa. In contrast, thickness of xylem wall, diameter of xylem vessels and the number of cells of the cortex of the differentiation root region significantly increased (64, 17, and 97%, respectively). The anatomical changes associated with low substrate PsiW indicate a net increase of root apoplatic paths; structures involved in water conduction increased their diameter under low substrate PsiW conditions and anatomical changes occurred during the first 24 h of water stress.