We show that the mammalian transcription Sp1 stimulates accurate transcription in a partially fractionated RNA polymerase II-dependent system from Drosophila cultured cells. Moreover, the extent of stimulation is equal for intact RNA polymerase II (polymerase IIA) and polymerase lacking the unique carboxyl-terminal domain of the largest subunit (polymerase IIB). We conclude that in this system Sp1 interacts with a component of the transcription machinery, other than the carboxyl-terminal domain, which is preserved between mammals and insects.