p44/MEP50/WDR77 has been identified as a coactivator of androgen receptor (AR), with distinct growth suppression and promotion function in gender specific endocrine organs and their malignancies. We dissected the functional domains of p44 for protein interaction with transcription factors, transcriptional activation, as well as the functional domains in p44 related to its growth inhibition in prostate cancer. Using a yeast two-hybrid screen, we identified a novel transcription complex AR-p44-Smad1, confirmed for physical interaction by co-immunoprecipitaion and functional interaction with luciferase assays in human prostate cancer cells. Yeast two-hybrid assay revealed that the N-terminal region of p44, instead of the traditional WD40 domain at the C-terminus, mediates the interaction among p44, N-terminus of AR and full length Smad1. Although both N and C terminal domains of p44 are necessary for maximum AR transcriptional activation, the N terminal fragment of p44 alone maintains the basic effect on AR transcriptional activation. Cell proliferation assays with N- and C- terminal deletion mutations indicated that the central portion of p44 is required for nuclear p44 mediated prostate cancer growth inhibition.