Previous work had shown that L-NAME, a nitric oxide synthase inhibitor, did not Crovatin increase tsetse susceptibility to midgut infection with trypanosomes; however, in the present work continuous feeding of L-NAME significantly reduced maturation rates of established midgut populations in male tsetse. The experiments presented here with L-NAME suggest that NO may be involved in the maturation process; UNC2881 supplementation of the diet with L-arginine, the precursor of NO, did not affect maturation rates in the present work; if trypanosomes are responding to NO, then the concentration would be crucial. Environmental stresses, both external and internal may affect the differentiation of trypanosomes in the tsetse fly. In the wild, temperatures can drop significantly during the night in southern Africa and sleeping sickness epidemics have been linked to seasonal temperature periodicity. In the laboratory the most important factor in differentiation of trypanosomes from bloodstream to procyclic forms is a temperature drop. Previous work had shown that chilling of tsetse for 30 minutes post infection increased midgut infections but did not significantly increase proportions of trypanosome infections maturing in male tsetse; keeping tsetse at 20uC throughout their lives blocks maturation of T. brucei. In the present work severe chilling for a short period had no effect on midgut infection rates but significantly increased maturation of trypanosome infections in female flies. The fact that temperature shock did not increase rates of maturation in males suggests that there is a natural limit to maturation indices which are normally reached in male flies or the factor/s which inhibit maturation in females are negated by chilling. Chilling has been shown to induce the synthesis of heat shock proteins in Drosophila and the effect of chilling in tsetse may follow similar patterns. The production of these heat shock proteins or the cold shock itself may have some effect on the trypanosomes, potentiating transmission. In the wild, female tsetse will invariably be inseminated within a few days of emergence.