Some T's in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (β-D-glucosyl-hydroxymethyluracil). In Leishmania about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA Polymerase II termination sites. Both this internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J is accompanied by massive read-through at RNA Polymerase II termination sites. The degree of read-through varies between transcription units, but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive read-through of transcriptional stops. We determined the exact location of base J in the genome of Leishmania by high-throughput sequencing of J containing DNA fragments. Samples were enriched for J-containing fragments by two independent methods: ChIP using an anti-J DNA antibody or by binding to a the J-binding protein JBP1. We studied the effect of loss of J on transcription using WT, JBP2 knockout (30-37% of WT level J), and JBP2 knockout cells grown in BrdU containing medium (13-16% of WT level of J). We used 6 RNA-seq libraries (three samples & two replica each) containing processed RNA products (transspliced and poly-adenylated) and 3 small RNA libraries representing the entire transcriptome.