One characteristic of cancer cells is the loss of this requirement, and such transformed cells gain the capacity to grow in an anchorage-independent manner. To simulate these conditions and assess this oncogenic characteristic, we cultured stable E2Fexpressing 3T3 lines in suspension in a semi-solid agarose medium. In this system, the non-transformed parental 3T3 cell line exhibited an almost complete requirement for attachment, as only a few small colonies were observed which did not show continuous growth when cultures were extended up to two months. Positive control H-Ras-transformed N57 cells generated a high frequency of large colonies that grew progressively over a one month period. The empty MINR1 vector-transduced 3T3 line showed a significant increase in the frequency of colony formation as compared to the parental line, but unlike the progressive growth of the N57 colonies, the MINR1-3T3 colonies were small, and had involuted by day 20. Ectopic expression of E2F1 and E2F6 in 3T3 cells had a relatively neutral effect on colony formation as compared to the empty vector-transduced line. In contrast, the E2F2- and E2F3-transduced 3T3 lines exhibited strong colony forming capacity. These cells generated colonies at frequencies approaching the H-Rastrans formed N57 cells, with individual colonies exhibiting strong, exponential growth over the entire one month culture period. Interestingly, ectopic expression of E2F4 and E2F5 in 3T3 fibroblasts resulted in frequencies of colony formation that were significantly lower than that of empty vectortransduced cells, and comparable to that of the non-transformed parental 3T3 line. Furthermore, the few colonies present in these cultures did not grow over time. These data suggest that E2F2 and E2F3 have strong oncogenic capacity, while E2F4 and E2F5 are anti-oncogenic in this system. E2F1 and E2F6 may be weakly oncogenic, but the background transforming capacity of the retroviral vector used in these studies makes our results with these two family members difficult to interpret. A number of genetic aberrations that promote cancer lead to deregulated E2F activity, including mutations in pRb, cyclinD1, p16INK4a and CDK4.