The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5′ end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5′ end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5′ ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5′ end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5′ end heterogeneity in combination with the 3′ end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.