The transforming potential of E2F1�C3 has been reported in various models and cell types, however, a systematic comparison of E2F1�C6 members has not been performed. To make a direct comparison of oncogenic function among these first six E2F family members, we have utilized a retroviral approach to generate stable lines of 3T3 fibroblasts specifically over-expressing have assessed the ability of these transgenic cell lines to grow under conditions of low serum, as well as to form colonies when suspended in soft agar. Our data demonstrates that E2F2 and E2F3 have strong pro-oncogenic capacity, whereas E2F4 and E2F5 are anti-oncogenic. E2F1 over-Cinepazide maleate expression was only variably achieved under these conditions, potentially due to high basal expression of endogenous E2F1 by Pheonix cells. We also had difficulty demonstrating E2F2 over-expression in these transient transfections, either due to low level expression of E2F2 protein, or relatively low sensitivity of the E2F2-specific antiserum. E2F-encoding retroviral supernatants Catharanthine produced from these Pheonix cell transfections were used to transduce non-transformed NIH 3T3 fibroblasts, and transductants were identified and purified by the expression of NGFR. Transduced 3T3 lines were expanded without selection, and stable NGFR expression was observed over several weeks in culture. We were able to detect specific over-expression of E2F2 through E2F6 in each respective 3T3 line under conditions of asynchronous growth, as compared to endogenous expression of these family members in an empty vector-transduced line. However, we were unable to detect over-expression of E2F1 in actively growing, E2F1-transduced 3T3 cells above that of the endogenous protein. To determine the effect of stable over-expression of individual E2F family members on asynchronous cell growth, we plated each E2Ftransduced line at low density in high serum medium, and enumerated the cells at 24 hour intervals over a four day culture period.The empty vector-transduced 3T3 line exhibited a consistent doubling rate of approximately 24 hours until reaching confluency between 72 and 96 hours. This pattern of growth closely resembled that of the parental, non-transduced 3T3 cells.