Vitellins from ovarian follicles and newly laid eggs of the stick insect Carausius morosus were examined by ion exchange chromatography on a HPLC Mono Q column. Under these conditions, vitellins from newly laid eggs resolved as two distinct peaks, referred to as VtA and VtB, that eluted at 8.5 and 12.0 min, respectively. On native gels, both VtA and VtB separated into two different variant forms (VtA´ and VtA´´, VtB´ and VtB´´). By two-dimensional gel electrophoresis, VtA´ and VtA´´ were shown to contain polypeptides A 1 , A 2 and A 3 . On the other hand, VtB´ and VtB" appeared to comprise polypeptides B 1 and B 2 and B 1 , A 1 , A 2 , B 2 and A 3 *, respectively. A similar Vt polypeptide composition was also observed by size-exclusion chromatography of vitellins from newly laid eggs. Vitellins from early vitellogenic ovarian follicles resolved into a single chromatographic peak at 7.5 min that coeluted with a major peak from the hemolymph of egglaying females. Ovarian follicles progressively more advanced in development exhibited a more complex chromatographic profile, consisting of three separate peaks. By two-dimensional gel immunoelectrophoresis, vitellins from ovarian follicles appeared to consist of two closely related, immunologically cross-reacting antigens that gradually shifted apart as ovarian development proceeded to completion. By size-exclusion chromatography, each Vt from ovarian follicles was shown to consist of a unique set of polypeptides different from those listed above. Single ovarian follicles were fractionated into yolk granules and yolk fluid ooplasm and tested by immunoblotting against Mab 12. Under these conditions, VtA variant forms in yolk granules and yolk fluid ooplasm reacted differently. Sections from ovarian follicles in different developmental stages were exposed to Mab 12 and stained with a peroxidase-conjugated, goat anti-mouse antibody. Regardless of