Determination of the most sensitive and susceptible organismal life stage is important for use of the organism in toxicity assessments of environmental pollutants. In the present study, the sensitivities and susceptibilities of larval developmental stages of two black fly sibling species, Simulium vittatum Zetterstedt cytospecies IS-7 and S. vittatum Zetterstedt cytospecies IIIL-1, were determined using the organophosphate-insecticide chlorpyrifos. Differences in sensitivity and susceptibility were determined through analysis of slopes and median lethal concentration values produced from 24-h orbital shaker toxicity tests, respectively. The results showed no difference in sensitivity or susceptibility between sibling species. However, early instar (second and third) as well as mid-instar (fourth and fifth) groupings of S. vittatum IS-7 were significantly more susceptible than late-instar (sixth and seventh) larval groupings. Likewise, mid-instar groupings of S. vittatum IIIL-1 were more susceptible than late-instar larval groupings. However, neither species showed significant differences in sensitivity among instar groupings. The results of the present study indicate that the mid-instar groupings of black fly larvae are the best choice for use in toxicity tests, both because of their increased susceptibility compared to late-instar groupings and because of their ease in handling and manipulating in the laboratory compared to early instar groupings.