Cytokinins may reflect soil water status and regulate rice (Oryza sativa L.) grain filling. This study investigated the changes in cytokinin levels in rice plants and their relations with grain filling under alternate wetting and drying irrigation. Two 'super' rice cultivars were field grown. Three irrigation regimes, alternate wetting and moderate soil drying (WMD), alternate wetting and severe soil drying (WSD), and conventional irrigation (CI, continuously flooded), were imposed after flowering. No significant differences in grain-filling rate, grain weight, and cytokinin content were observed for the earlier-flowering superior spikelets among the three irrigation regimes. For the later-flowering inferior spikelets, however, their grain-filling rate and grain weight were significantly increased in the WMD and significantly reduced in the WSD when compared with those in the CI. Cytokinin contents in shoots (inferior spikelets and the flag leaves) in the WMD at the soil drying time were comparable with those in the CI, but they were significantly increased when plants were rewatered. The WSD significantly reduced cytokinin contents in the shoot either during soil drying or during the rewatering period. Cytokinin contents in roots showed no significant difference between the WMD and CI regimes. The WSD increased trans-zeatin-type cytokinins, whereas it reduced isopentenyladenine-type cytokinins, in roots. Grain-filling rate and grain weight of inferior spikelets were very significantly correlated with cytokinin contents in these spikelets. The results suggest that a post-anthesis WMD holds great promise to improve grain filling of inferior spikelets through elevating cytokinin levels in the rice shoot.