A nuclear DNA complex containing DNA polymerase and SV40 T-antigen was isolated from nuclei of SV40-transformed mouse fibroblasts. DNA polymerase could be separated from the complex. The remaining DNA/T-antigen-containing complex stimulated DNA polymerase alpha activity about 10-fold. The complex contained 4 major proteins with molecular weights of 46, 54, 76, and 94 kilo-dalton (KD). The stimulation activity was retained by protein A-Sepharose loaded with specific IgG from SV40-tumor bearer serum, or from antisera against the 94 KD and 76 KD components and was partially inhibited in the presence of these antisera. The stimulation activity was completely abolished by treatment of the complex with trypsin or DNase I.