African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.Trypanosoma brucei, a unicellular parasite which causes human sleeping sickness, is transmitted between mammals by the tsetse fly. Adaptation of the parasite to the mammalian host and the fly vector entails distinct life cycle stages which differ considerably in morphology, surface coat composition, energy metabolism, and proliferation status (reviewed in reference 23). In the bloodstream and tissue fluids of the mammalian host, T. brucei proliferates as a long slender form and differentiates into a growth-arrested short stumpy form that is preadapted for survival in the fly. When bloodstream forms are taken up during a blood meal by the insect vector, the stumpy form differentiates rapidly to the procyclic (insect) form in the lumen of the fly midgut. The parasite continues its life cycle by progressing through a series of further developmental stages culminating, in the salivary glands of the fly, in differentiation to the metacyclic form which is infective for a new mammalian host (44).