AbstractThe physiological control of stomatal opening by which plants adjust for water availability has been extensively researched. However, the impact of water availability on stomatal development has not received as much attention, especially for amphistomatic plants. Therefore, the acclimation of stomatal development in basil (Ocimum basilicumL.) leaves was investigated. Our results show that leaves developed under water-deficit conditions possess higher stomatal densities and decreased stomatal length for both the adaxial and abaxial leaf sides. Although the stomatal developmental reaction to water deficit was similar for the two leaf surfaces, it was proven that adaxial stomata are more sensitive to water stress than abaxial stomata, with more closed adaxial stomata under water-deficit conditions. Furthermore, plants with leaves containing smaller stomata at higher densities possessed a higher water use efficiency. Our findings highlight the importance of stomatal development as a tool for long-term acclimation to limit water loss, with minimal reduction in biomass production. This highlights the central role that stomata play in both the short (opening) and long-term (development) reaction of plants to water availability, making them key tools for efficient resource use and anticipation of future environmental changes.