Leaves possess intrinsic information about their final size, but the developmental mechanisms setting the limits of growth are not well characterized. By screening enhancer trap lines that show a specific expression pattern in leaf primordia, we isolated one line, 576. This line contains a T-DNA insertion upstream of the basic helix–loop–helix (bHLH) transcription factor SPATULA (SPT) gene, and shows expression in the basal region of young leaves, where cell proliferation is active. An spt loss-of-function mutation increased leaf size and total cell number within a leaf, while SPT overexpression decreased leaf size and total cell number within a leaf. Although spt mutations did not affect cell size, SPT overexpression decreased the cell size in fully expanded leaves. Genetic analysis suggested that SPT acts independently from another set of cell proliferation-dependent organ size regulators ANGUSTIFOLIA3 (AN3) and GROWTH REGULATING FACTOR5 (AtGRF5). Detailed analysis of spt leaf development showed that the spt mutation enlarged the size of the meristematic region in leaf primordia, while overexpression of AtGRF5 promoted cell proliferation without affecting the enlargement of the meristematic region. These results suggest that SPT functions as a repressor of leaf growth and that meristematic region size in young leaf primordia, in terms of proliferative cell number within leaf primordia, is another target of leaf size determination, which previously had not been identified. [ABSTRACT FROM PUBLISHER]