The cytokinin activity in barley (Hordeum vulgare L.) caryopses (determined by the soybean [Glycine max (L.) Merr.] callus assay method) decreases very rapidly after pollination. Small caryopses, whether genetically determined, resulting from de‐awning of spikes, or from damage by high water content of soil after pollination, matured earlier and had lower cytokinin activity. Differences in cytokinin content were detected earlier than those in grain size. The results suggest that plant growth substancs such as cytokinins participate in the regulation of grain size, possibly by influencing accumulation processes and the duration of the filling period. It is probable that roots also play a role in regulating grain size by supplying the spikes with plant hormones.