The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA-recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152-155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181-359). Consistently, point mutations in an evolutionarily conserved motif (aa 202-205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.