In nutrient-deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane-bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell-cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole-arrested pseudo-prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)-kinase inhibitors wortmannin, LY294002 and 3-methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1–/– embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3-kinases or other wortmannin/LY294002-sensitive kinases in mitotic membrane traffic regulation.