The yeast pet18 mutant exhibits three distinct phenotypes: temperature-sensitive lethality, failure to maintain a dsRNA virus, and respiration deficiency. We have isolated a yeast mutant, H53, with phenotypes identical to those of pet18. Based on PCR and Southern hybridization analysis, H53 was found to result from a large chromosomal deletion extending from YCR019w to YCR028c on chromosome III. Genetic analysis was carried out on H53 to correlate individual loci with each of the observed phenotypes. Disruption of YCR020c-a/MAK31 brought about a loss of dsRNA without affecting the temperature sensitive phenotype. The loss of YCR020w-b/HTL1, which encodes a hypothetical protein of 78 amino acids in length, was shown to be responsible for the temperature-sensitive lethality of the H53 mutant. Using immunoblotting, we demonstrated that a 7-kDa protein was indeed expressed in wild-type yeast, but not in a HTL1 deletion mutant. Moreover, the significance of HTL1 was investigated by isolating genes that are functionally associated with HTL1. We demonstrated that Rsc8p interacts physically with Htl1p, and that the genes RSC3, STH1 and RSC30 interact with HTL1. Thus, HTL1 may play a role in the function of the RSC complex.