We have previously reported the construction and characterization of an autonomously replicating plasmid in Trypanosoma brucei. In this plasmid the procyclic acidic repetitive protein (PARP) gene promoter drives the transcription of a selectable marker. Deletion of this promoter incapacitates the plasmid, suggesting its utilization as a promoter-trap. Three independent libraries were created by inserting variously digested T.brucei genomic DNA into this promoterless construct. Transfection of these libraries into procyclic T.brucei and the subsequent isolation of episomes led only to the reisolation of the PARP promoter. Additionally, a ribosomal RNA promoter failed to keep the construct as an episome, although it can sustain mRNA transcription in T.brucei and was shown to be an efficient promoter in this construct. Finally, by using a transient replication assay involving the methylation-sensitive restriction endonuclease DpnI to distinguish between input and replicated DNA, we showed that the PARP promoter-bearing construct could replicate autonomously in procyclic T.brucei, but the corresponding construct with the rRNA promoter could not. The close association between elements that sustain transcription and DNA replication in T.brucei mirrors results observed in several higher eukaryotes and their viruses and suggests an ancient origin of this feature.