African trypanosomes show monoallelic expression of one of about 20 telomeric variant surface glycoprotein (VSG) gene-expression sites (ESs) while multiplying in the mammalian bloodstream. We screened for genes involved in ES silencing using flow cytometry and RNA interference (RNAi). We show that a novel member of the ISWI family of SWI2/SNF2-related chromatin-remodelling proteins (TbISWI) is involved in ES downregulation in Trypanosoma brucei. TbISWI has an atypical protein architecture for an ISWI, as it lacks characteristic SANT domains. Depletion of TbISWI by RNAi leads to 30-60-fold derepression of ESs in bloodstream-form T. brucei, and 10-17-fold derepression in insect form T. brucei. We show that although blocking synthesis of TbISWI leads to derepression of silent VSG ES promoters, this does not lead to fully processive transcription of silent ESs, or an increase in ES-activation rates. VSG ES activation in African trypanosomes therefore appears to be a multistep process, whereby an increase in transcription from a silent ES promoter is necessary but not sufficient for full ES activation.