2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates have been investigated as substrates for T7 RNA polymerase. Michaelis-Menten kinetic parameters are reported for the incorporation of 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, and 2'-amino-2'-deoxyuridine into runoff transcripts. The 2'-amino derivative of uridine is a better substrate than the 2'-fluoro derivative. Gel electrophoretic analysis shows that full-length transcripts with a length of 2500 nucleotides can be obtained with the analogues, although a considerable amount of shorter fragments accompanies the full-length product. In keeping with the kinetic analysis, the 2'-aminouridine triphosphate gives a cleaner product than the 2'-fluoro analogue. Transcription of two tRNA genes shows that such shorter templates can be transcribed to full-length products essentially without premature termination with any of the analogues.