The three-dimensional architecture of a quartet of microtubules (QM) of Trypanosoma evansi and its related structures was examined with a transmission electron microscope (TEM). The initiation of the QM as a successive bundle of microtubules was confirmed at the juxtaposition of the second barren body (SBB). After connecting with the distal part of the basal body (BB), the QM ascended to the level of the terminal plate (TP) and gained access most closely to the inner membrane of the flagellar pocket (FP) and formed the special structure together with macula adherens (MA) and the electron dense linear substance (EDLS); then it follows its way around the FP from the posterior to the anterior and fills the subpellicular space at the underneath of the flagellum. This special structure disappeared when the associated-flagellum became the extracellular flagellum (EF), where the QM intervened into the row of the subpellicular microtubules (SM). This EDLS is suggested to represent a demarcating line (DL). In the deep cytoplasm of the flagellar root, connections via several microtubules between the BB and the SBB and via electron zonal substance (EDZS) between the BB and the mitochondrion (M) were observed. As a result, a connecting system among the flagellar apparatus and its accessory structures seems probable.