Vps34, the sole PtdIns 3-kinase in yeast, is essential for autophagy. Here, we show that the lipid-kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3) P. The protein-kinase activity of Vps15, a regulatory subunit of the PtdIns 3-kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3) P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3) P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3) P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3) P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3) P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3) P in autophagy.