Splicing factor, proline- and glutamine-rich protein (SFPQ), was identified in eight human cultivated cell lines by proteomic approaches. The cell proteins have been separated by means of two-dimensional gel electrophoresis in two modifications and identified by matrix-assisted laser desorption ionization mass spectrometry with further tandem mass spectrometry. The analysis of proteins from three human sarcomas cell lines (RD, U-2 OS and SK-UT-1B), three human renal adenocarcinomas cell lines (A-498, 769-P and OKP-GS), and two prostate adenocarcinomas cell lines (DU-145 and PC-3) revealed several electrophoretic isoforms of SFPQ protein. Differences between theoretical and experimental molecular masses and isoelectric points of SFPQ protein have been observed. Detailed investigation of SFPQ peptides by tandem mass spectrometry has detected new phosphorylation state of threonine residue in 168 position of SFPQ isoform in rhabdomyosarcoma cell line. Furthermore, SFPQ has not been identified during proteomic study of several nonmalignant cell lines, including cultured human mesenchymal stromal cells and myoblasts. However, SFPQ has been found in all malignant cell lines in high quantity. In particular, its fractions are abundant in sarcomas cell lines as opposed to nonmalignant mesenchymal cells. It is assumed that high quantity of SFPQ in sarcomas cell lines may affect tumorigenesis.