We have established conditions for Trypanosoma brucei permeable cells to study transcription and trans-splicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCl, whereas higher potassium concentrations were inhibitory. In addition, MgCl2 concentrations above 3 mM led to the accumulation of a 3' end shortened SL RNA species, which has been previously reported not to participate in trans-splicing. Using conditions optimal for the synthesis of the SL RNA, we observed accurate trans-splicing of newly-synthesized alpha-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced alpha-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.