As a step toward determining the metabolic role(s) of a 5'----3' exoribonuclease (XRN1), a yeast gene, XRN1, encoding XRN1, was first cloned, then disrupted to test its essentially or effect on yeast cell growth. Clones in the high-copy-number plasmid YEp24 cause overproduction (fivefold) of XRN1 in yeast cells, as measured by either poly(A) hydrolytic activity or immunoreactivity. Restriction mapping and deletion analysis showed that the XRN1 gene is located on a 6.7-kb XbaI-XhoI fragment of chromosome VII. The normal gene was disrupted in two haploid yeast strains by integrating a fragment with a BglII-deleted segment replaced with the yeast URA3 gene, and the disrupted strains lack XRN1. Successful transformation of haploid cells showed that the gene is not essential, but its absence markedly affected the cell growth rate. The growth defect is corrected by introduction of the XRN1 gene on a plasmid back into the disrupted yeast.