Trypanosoma brucei can undergo antigenic variation by switching between distinct telomeric variant surface glycoprotein gene (vsg) expression sites (ESs) or by replacing the active vsg. DNA rearrangements have often been associated with ES switching, but it is unclear if such rearrangements are necessary or whether ES inactivation always accompanies ES activation. To explore these issues, we derived ten independent clones, from the same parent, that had undergone a similar vsg activation event. This was achieved in the absence of an immune response, in vitro, using cells with selectable markers integrated into an ES. Nine of the ten clones had undergone ES switching. Such heritable changes in transcription state occurred at a frequency of approximately 6 x 10(-7). Comparison of switched and un-switched clones highlighted the dynamic nature of T. brucei telomeres, but changes in telomere length were not specifically associated with ES switching. Mapping within and beyond the ESs revealed no detectable DNA rearrangements, indicating that rearrangements are not necessary for ES activation/inactivation. Examination of individual cells indicated that ES activation consistently accompanied inactivation of the previously active ES. In some cases, however, we found cells that appeared to have efficiently established the switched state but which subsequently, at a frequency of approximately 2 x 10(-3), generated cells expressing both pre- and post-switch vsgs. These results show that ES activation/inactivation is usually a coupled process but that cells can inherit a propensity to uncouple these events.