ABSTRACT The flagellum of Euglena gracilis has been studied comparatively, using the freeze-fracture technique and with thin sections involving a tannic acid staining procedure. The flagellar membrane exhibits specializations in the form of two groups of intramembrane particle rows (IMP rows; mean IMP diameter, 6 nm) and single clusters of larger IMPs (mean IMP diameter, 10 nm) associated with the edge of one of the two groups of IMP rows. The IMP rows are helically arranged. The two groups of IMP rows are separated from each other by two membrane areas (measuring 170 and 300 nm in length) devoid of IMP arrays. The IMP rows are arranged in a four-start helix around the flagellum. A comparative morphometric analysis of IMP arrays and the arrangement of the two types of non-tubular mastigonemes (evaluated from tannic-acid-stained thin sections) reveals that the two groups of IMP rows correspond to the attachment points of the short non-tubular mastigonemes, whereas the cluster of three to seven larger IMPs corresponds to the attachment points of a group of the long non-tubular mastigonemes. Flagellar membrane specializations also occur with respect to sterols, since filipin-sterol complexes are absent from all plasma membrane regions except the flagellar membrane. On the flagellar membrane filipin-sterol complexes occur predominantly in a membrane area free of IMP-arrays and are probably related in location to a prominent microtubule-membrane linkage. Details of the linkage between axonemal microtubules and the flagellar membrane have been evaluated, individual microtubule doublets have been numbered and the absolute configuration of the Euglena flagellum has been established. In the reservoir region the flagellar membrane of the emergent flagellum is not covered by mastigonemes, but exhibits IMP arrays in the form of IMP rows and IMP clusters. In contrast to specializations on the free part of the emergent flagellum, in the reservoir region IMP rows are not regularly spaced, are distributed on opposite membrane halves (in the reservoir region on the E-face, in the free flagellum on the P-face) and often not associated closely with the clusters of larger IMPs. An hypothesis is presented explaining how mastigonemes might attach to the flagellar membrane and finally become oriented in their characteristic arrays. It is suggested that the observed IMP specializations in the flagellar membrane are (a) responsible for attachment of mastigonemes to the flagellar membrane, (b) responsible for linkage of mastigonemes to intraflagellar structures associated with axonemal microtubules, and (c) play an important role as a template for the development of the characteristic mastigoneme arrays.