The MKT1 gene was defined by recessive alleles present in many laboratory strains of Saccharomyces cerevisiae that result in loss of M2 double-stranded RNA at temperatures above 30 degrees C if L-A-HN double-stranded RNA is present but not if L-A-H is present. I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2. The gene location was defined by deletions, and a 2.8-kilobase transcript corresponding to the gene was detected. The recessive natural-variant mutations are not deletions as judged by Southern blots, but deletions of the MKT1 gene constructed in vitro and used to replace the normal gene surprisingly resulted in the same phenotype as that of the mkt1 natural variants, namely, a temperature-dependent maintenance of M2 double-stranded RNA. Thus the MKT1 gene product is only needed for M2 replication or maintenance at temperatures above 30 degrees C and if L-A-HN is present. The temperature dependence does not reflect the thermolability of a mutant gene product, as had previously been thought, nor does L-A double-stranded RNA need MKT1, as previously hypothesized. MKT1 may be involved in the process of packaging M2 double-stranded RNA. MKT1 is dispensable for host cell growth, mating, meiosis, and spore germination.