Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.