The Knr4 protein, known to be involved in the regulation of cell wall assembly in Saccharomyces cerevisiae, strongly interacts with the tyrosine tRNA synthetase protein encoded by TYS1 as demonstrated by the genetic two-hybrid system and a biochemical pull-down experiment using GST--Tys1p fusion. Data reported here raise the possibility that this physical interaction between these proteins is required for dityrosine formation during the sporulation process. In addition, it is shown that the efficiency of spores formation was drastically reduced in diploid cells homozygous for the disruption of KNR4 or for a temperature-sensitive mutation of TYS1, although this effect could be independent of their protein interaction. Altogether, these data provide novel functions of Knr4p and Tys1p to those that were known before.