Mammalian transcription factor IIF (TFIIF) is a heterodimer composed of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits. TFIIF has been shown to bind RNA polymerase II and control the activity of the enzyme in both the initiation and elongation stages of transcription. Although previous studies have established a role for RAP30 in assembly of the preinitiation complex and in transcription initiation, information on the function of RAP74 in these processes has been lacking. Using a highly purified transcription system and assays that permit sensitive measurement of the contributions of both RAP30 and RAP74 to TFIIF function, we have investigated the roles of these TFIIF subunits in transcription initiation and elongation. Results of template competition experiments indicate that both RAP30 and RAP74 contribute to the formation of stable preinitiation intermediates containing RNA polymerase II. Investigation of the role of TFIIF in transcription initiation indicates that both RAP30 and RAP74 function in synthesis of the first few phosphodiester bonds of nascent transcripts and in formation of Sarkosyl-resistant pre-initiation intermediates. Finally, kinetic experiments indicate that both RAP30 and RAP74 function in TFIIF-mediated stimulation of the rate of RNA chain elongation by RNA polymerase II.