Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1alpha (EF1alpha), was first described to be essential for the G1 to S phase transition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1alpha-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.