A series of experiments was designed to investigate the activity of the SV40 early promoter and enhancer in rat pituitary tumor cells in culture but still yet not clear so, a series of enhancer promoter-reporter plasmids were constructed to study the SV40 activity in the rat pituitary tumor cell lines (GH3 and GC) then sub-cloned into pSV2(enhancer-minus)-CAT, then purified in gel and ligated into pRSV(enhancer-minus)-CAT (Sph1 restricted) then transformed into E.Coli then make restriction analysis. From the results we observed that SV40 early promoter can be activated by heterogeneous enhancers in pituitary tumor cells.