Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI7, W/2, and UN3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, U/3/4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UN4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4lUBl4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rap idly, apparently a novel phenotype in yeast. Furthermore, ubirllubirl diploids are sporulation-defective. ubi4 mutants an? also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the US14 gene is similarly induced by either heat stress or starvation. These results indicate that 0814 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.