E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle. The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain. For E2F-1, E2F-2, and E2F-3, the repressor is the product of the retinoblastoma gene, pRb. E2f-4 interacts with pRb-related p107 and not with pRb itself. Recently, a cDNA encoding a third member of the retinoblastoma gene family, p130, was isolated. p130 also interacts with E2F DNA binding activity, primarily in the G0 phase of the cell cycle. We report here the cloning of a fifth member of the E2F gene family. The human E2F-5 cDNA encodes a 346-amino-acid protein with a predicted molecular mass of 38 kDa. E2F-5 is more closely related to E2F-4 (78% similarity) than to E2F-1 (57% similarity). E2F-5 resembles the other E2Fs in that it binds to a consensus E2F site in a cooperative fashion with DP-1. By using a specific E2F-5 antiserum, we found that under physiological conditions, E2F-5 interacts preferentially with p130.