Saccharomyces cerevisiae vacuole inheritance requires two l ow m olecular weight a ctivities, LMA1 and LMA2. LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (I 2 B ). Here we show that the second low molecular weight activity (LMA2) is monomeric I 2 B . Though LMA2/I 2 B was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: ( i ) Low M r protease B inhibitors cannot substitute for LMA1 or LMA 2, ( ii ) LMA1 and LMA2 promote the fusion of vacuoles from a strain that has no protease B, ( iii ) low concentrations of LMA2 that fully inhibit protease B do not promote vacuole fusion, and ( iv ) LMA1, in which I 2 B is complexed with thioredoxin, is far more active than LMA2/I 2 B in promoting vacuole fusion and far less active in inhibiting protease B. These studies establish a new function for I 2 B .