Mastermind (Mam) is one of the evolutionarily conserved elements of Notch signaling. Genetic analyses in Drosophila implicated it as an important positive regulator of the pathway. We show here identification of two new members of human Mam family (human Mastermind-2 (hMam-2) and human Mastermind-3 (hMam-3)), which retain characteristics similar to human Mastermind-1 (hMam-1) and Drosophila Mastermind. Both hMam-2 and hMam-3 stabilize and participate in the DNA-binding complex RBP-J/CBF-1 protein and the Notch intracellular domains that serve as intermediates of the signaling. Both hMam-2 and hMam-3 enhanced the activation of transcription from a target promoter by Notch signaling. However, we also show evidence that the activation of the target promoter by Notch3 and Notch4 is more efficiently potentiated by hMam-2 than by hMam-1 or -3. The multiplicity of Mam proteins in the mammalian system may help provide divergence to the strength of the Notch signals in different cell types.