The cell cycle is under the control of various positive and negative regulators. Two such regulators are the E2F family of transcription factors and the p53 tumor suppressor protein. While E2F proteins are implicated in promoting the S phase of the cell cycle, p53 has the potential to arrest cells in G1 phase and thereby prevent entry into S phase. Because they perform seemingly opposite functions in the control of cell growth, a possibility of functional interactions between E2F and p53 was investigated. It was found that p53 specifically inhibited activated transcription by E2F-5 but not by E2F-1. Investigation into the mechanism of action established that heterodimer formation and the DNA-binding steps were not significantly inhibited by p53. However, the transcriptional activation step of E2F-5 activity, as examined by using a Gal4 DNA-binding domain chimera, was specifically inhibited by p53. Interestingly, p53 could also inhibit transcriptional activation by E2F-4 but not by E2F-2 or E2F-3. The results indicate that p53 differentially regulates the activities of two subclasses (E2F-1/-2/-3 vs. E2F-4/-5) of E2F transcription factors. Detailed analysis using a two-hybrid approach in mammalian cells indicated lack of physical interaction between p53 and E2F-5, DP-1, or E2F-1. Reciprocal analysis revealed that whereas E2F-1 dramatically inhibited p53-activated transcription, E2F-5 or DP-1 did not. Thus, nonreciprocal functional interactions exist between various members of the E2F family of transcription factors and p53 tumor suppressor protein. The complex interplay between various positive and negative regulators of cell growth, such as E2F and p53 proteins, may be crucial in determining the ultimate outcome in terms of cell cycle arrest, cell growth, or apoptosis.