DNA-topoisomerase I has been implied in RNA splicing because it catalyzes RNA strand transfer and activates serine/arginine-rich RNA-splicing factors by phosphorylation. Here, we demonstrate a direct interaction between topoisomerase I and pyrimidine tract binding protein-associated splicing factor (PSF), a cofactor of RNA splicing, which forms heterodimers with its smaller homolog, the nuclear RNA-binding protein of 54 kDa (p54(nrb)). Topoisomerase I, PSF, and p54(nrb) copurifled in a 1:1:1 ratio from human A431 cell nuclear extracts. Specific binding of topoisomerase I to PSF (but not p54(nrb)) was demonstrated by coimmunoprecipitation and by far Western blotting, in which renatured blots were probed with biotinylated topoisomerase I. Chemical cross-linking of pure topoisomerase I revealed monomeric, dimeric, and trimeric enzyme forms, whereas in the presence of PSF/p54(nrb) the enzyme was cross-linked into complexes larger than homotrimers. When topoisomerase I was complexed with PSF/p54(nrb) it was 16- fold more active than the pure enzyme, which could be stimulated 5- and 16- fold by the addition of recombinant PSF or native PSF/p54(nrb), respectively. A physiological role of this stimulatory mechanism seems feasible, because topoisomerase I and PSF showed a patched colocalization in A431 cell nuclei, which varied with cell cycle.