The transcription factor PU.1 plays an important role in the development of the myeloid and lymphoid lineages and regulates the transcription of several genes expressed in these cells. Ser41 is conserved in the acidic region (33-47) of PU.1 from a variety of eukaryocytes and has been reported to be one of the two important Ser residues (S41 and S45) for the function of PU.1. In the present study, however, we found that rat PU.1 has Gly at position 41. To elucidate the role of amino acid residues at 41 and 45 in functions of PU.1, we generated mutants of rat PU.1, G41S, G41A, and S45A, and analyzed their transcription-enhancing activities by using two different systems, transient reporter assay system and retroviral transfection system. The amino acid substitution at 41 of PU.1 causes no effect on both transcription-enhancing activity for M-CSF receptor promoter and the cooperative transcription-enhancing activity with GATA-1 for FcRI alpha-chain promoter. Furthermore, the substitution at 41 also had no effect on the activity to induce monocyte-specific gene expression in the bone marrow-derived hematopoietic cells. From these results, we conclude that Ser41 as well as Ser45 are not essential for the promoter-upregulating function of PU.1.