Cloned Drosophila melanogaster tRNA genes have been transcribed in a homologous cell-free extract isolated from a Schneider II cell line. The major product of the reaction is a tRNA precursor which is processed to a tRNA sized species. The kinetics of transcription has been followed for 5 different valine tRNA gene clones. The results demonstrate formation of stable transcription complex with at least two kinetic steps. While the rate of formation of the transcription complex is similar to different clones, the ultimate rate of transcription varies dramatically. Comparison of the DNA sequence of the tRNA genes suggests that rate determining nucleotides lie outside the canonical tRNA split-internal promoters.