The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.