Microautophagy is the uptake of cytosolic compounds by direct invagination of the vacuolar/lysosomal membrane. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. Autophagic tubes are topologically equivalent to the invaginations at multivesicular endosomes. At the tip of an autophagic tube, vesicles (autophagic bodies) pinch off into the vacuolar lumen for degradation. In this study we have identified calmodulin (Cmd1p) as necessary for microautophagy. Temperature-sensitive mutants for Cmd1p displayed reduced frequencies of vacuolar tube formation and/or abnormal tube morphologies. Microautophagic vacuole invagination was sensitive to Cmd1p antagonists as well as to antibodies to Cmd1p. cmd1 mutants with substitutions in the Ca2+-binding domains showed full invagination activity, and vacuolar membrane invagination was independent of the free Ca2+ concentration. Thus, rather than acting as a calcium-triggered switch, Cmd1p has a constitutive Ca2+-independent role in the formation of autophagic tubes. Kinetic analysis indicates that calmodulin is required for autophagic tube formation rather than for the final scission of vesicles from the tip of the tube.