Transcription of the multicopyβ-tubulin locus inLeishmania donovani promastigotes was examined by nucleic acid hybridization techniques. By northern analysis of promastigote RNA multipleβ-tubulin mRNAs were detected. The major species of 2.2 kb RNA is derived from the tandem repeat cluster ofβ-tubulin genes, the other two (2.4 and 2.6 kb) are presumably derived from dispersed genomic loci. Combined S1-nuclease and primer extension mapping experiments demonstrated the presence of a single 5′-terminus with a 35 nucleotide spliced-leader sequence. The 3′-termini are heterogeneous. The development of a nuclear run-on system inLeishmania for studying transcription of individual genes is reported. Active but transient RNA polymerase II activity was observed in this system. Using specific DNA probes for labelled run-on RNA it was shown thatβ-tubulin transcription occurs asymmetrically (i.e., on one strand of the DNA template) in anα-amanitin sensitive manner. The significance of these results for the life cycle of the parasite is discussed.