A temperature-sensitive mutant of rat fibroblast line 3Y1, 3Y1tsD123, is arrested in the G1 phase of the cell cycle at restrictive temperature. Using a human cDNA expression library, we succeeded in the molecular cloning of a cDNA (designated as D123 cDNA) that functionally complemented the temperature-sensitive mutation. We then isolated rat D123 cDNA from a 3Y1 cDNA library using the human D123 cDNA probe. Both human and rat D123 cDNA contained a long open-reading-frame coding for a novel protein (designated as D123 protein) of 336 amino acids. A comparison of the nucleotide sequences of rat D123 cDNA and the corresponding cDNA of 3Y1tsD123-obtained by polymerase chain reaction in conjunction with reverse transcription showed that there was a difference in a single nucleotide that must cause a single amino acid change in D123 protein. Antibody raised against the recombinant protein of human D123 cDNA detected a 44-kDa D123 protein in human embryo lung fibroblasts and 3Y1. The level of D123 protein was much lower in 3Y1tsD123 than in 3Y1 at both permissive (33.8 degrees C) and restrictive (39.8 degrees C) temperatures. We suppose that quantitative and qualitative changes of D123 protein due to the amino acid change cause the temperature-sensitive arrest of 3Y1tsD123. On the other hand, the level of D123 protein in 3Y1 did not change significantly whether cells were proliferating or arrested at the saturation cell density. This indicates that the G1 arrest of 3Y1 at the saturation cell density does not involve the control of D123 protein level.