The simian virus 40 (SV40) DNA fragment encoding small t antigen was cloned with expression vector pUC8 for the purification of small t-antigen. Small t antigen produced in Escherichia coli was a hybrid protein of 22,000 daltons which comprised about 6% of the total protein. Hybrid small t antigen maintained antigenic activity which was examined by Western-blotting and enzyme-linked immunoassay. Hybrid small t antigen was extracted from E. coli with urea and purified from a small t-antigen band in SDS-polyacrylamide gel. The purified antigen maintained antigenic activity.