The multilayered structure (MLS), a component of the locomotory complex of plant sperm, has been utilized extensively by taxonomists in establishing phylogenetic relationships between the lower plants and algae. Unfortunately, there has been almost no biochemical characterization of the MLS and, in those studies that did attempt a characterization, conflicting results were obtained. We utilized antisera to the calcium-binding protein centrin to probe thin sections of the mid-stage spermatids of the anthocerote Phaeoceros laevis, the hepatic Sphaerocarpos texanus, and the pteridophyte Ceratopteris richardii embedded in L. R. White resin. The lamellar strip (LS; layers S2-S4) of the MLS in each of these species is labelled strongly with anti-centrin, but the S1 layer, composed of microtubules, is not. In Ceratopteris, centrin is also detected in the amorphous electron opaque material that connects the basal bodies of the flagella. Both the MLS and the amorphous zones are putative microtubule organizing centers. Extracts from axenic cultures of Ceratopteris subjected for electrophoresis and Western blotting revealed a reactive band at 19.3 kDa, a protein similar in molecular mass to algal centrin. These data indicate that the MLS is composed at least partially of the protein centrin or a protein antigenically-related to centrin. This report is the first electron microscopic immunocytochemical demonstration that a centrin homologue is found in land plants and that it occurs at putative microtubule organizing centers.