Trypanosoma brucei evades the mammalian immune response by a process of antigenic variation. This involves mutually exclusive and alternating expression of telomere-proximal variant surface glycoprotein genes (vsgs), which is controlled at the level of transcription. To examine transcription repression in T. brucei we inserted reporter genes, under the control of either rRNA or vsg expression site (ES) promoters, into various chromosomal loci. Position-dependent repression of both promoters was observed in the mammalian stage of the life cycle (bloodstream forms). Repression of promoters inserted into a silent ES was more pronounced closer to the telomere and was bi-directional. Transcription from both ES and rRNA promoters was also efficiently repressed at a non-telomeric vsg locus in bloodstream-form trypanosomes. In cultured tsetse fly midgut-stage (procyclic) trypanosomes, in which vsg is not normally expressed, all inserted rRNA promoters were derepressed but ES promoters remained silent. Our results suggest that vsg promoters and ectopic rRNA promoters in bloodstream-form T. brucei are restrained by position effects related to their proximity to vsgs or other features of the ES. Sequences present in rRNA promoters but absent from vsg ES promoters appear to be responsible for rRNA promoter-specific derepression in procyclic cells.