The fluorescence recovery after photobleaching (FRAP) method, in combination with confocal laser scanning microscopy, represents one of the basic approaches to studying the properties of proteins in living mammalian cells. However, the data of different authors on the dynamic properties of the same protein and even in cells of the same type can differ greatly. Until now, the reasons for such discrepancies have not been specifically analyzed. In the present work, using the example of nucleolar protein fibrillarin fused with EGFP, we studied the effect of the area of the irradiated region (the region of interest (ROI)) and temperature conditions of experiments on the main dynamic characteristics of the protein-the portion of the mobile fraction of protein and the half-recovery time of fluorescence after photobleaching (t1/2). The obtained results have shown that both parameters affect markedly the estimation of the fibrillarin-EGFP mobility in HeLa cells. It was concluded that, in FRAP experiments the ROI area can be standardized and, where possible, minimized. In addition, when analyzing the dynamic characteristics of the nucleolar proteins, which participate in the temperature-dependent enzymatic reactions, it is necessary to maintain standard temperature conditions. © 2012 Pleiades Publishing, Ltd.