A simian virus 40 (SV 40) DNA fragment encoding small t antigen was cloned in expression vector pUC8 for the purification of the antigen. The SV40 Hind III B fragment was inserted into the Hind III site of pUC8. A plasmid having the lacZ' and small t antigen genes in the same orientation was designated as pSVt. pSVt encodes the entire small t antigen and an extra 18 amino acids at the amino terminus of the antigen. E. coli transformed with pSVt produced hybrid small t antigen (22 kDa) which comprised about 6% of the total protein. The hybrid small t antigen reacted in immunoblot analysis with SV40-induced tumor-bearing hamster serum. Hybrid small t antigen was extracted from E. coli, and purified by preparative SDS-PAGE. The antigen was extracted from the gel using formic acid solution with high-yield. The gel-purified antigen showed the same antigenic reactivity as crude antigen.