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Summary. Ecdysteroid titers were measured in whole-body extracts of pedogenetically reproducing larvae of the dipteran insect Heteropeza pygmaea and in the dietary fungus. The titers are very low in the first 3 days of larval growth, but increase during the last 2 days. The level of 20-hydroxyecdysone is then significantly higher than that of ecdysone. Measurements of the titers in the fungus gave no conclusive results.Key words. Ecdysteroids, pedogenesis; Heteropeza pygmaea, mycelium.Heteropeza pygmaea, like many other gall midges, is phytophagous and feeds on fungus. Fresh (3-day-old) mycelium causes the H.pygmaea larvae to reproduce by pedogenesis (= parthenogenetic reproduction in the larval stage3.4). In pedogenetic reproduction the ovaries will develop prematurely and produce follicles already in the young larvae within the first 24 h of larval development 5. When H.pygmaea larvae are set on aged (7-day-old) mycelium, the mode of reproduction is altered; ovarian development is retarded and follicle formation only takes place after several days 3' 5. The effect of 20-hydroxyecdysone on ovarian development of H.pygmaea was examined in an earlier investigation 6. The test system consisted of larval ovaries cultured in hemolymph drops. In this in vitro system the hormone was shown to stimulate strongly formation and separation of pedogenetically developing follicles. The same result was obtained when high doses of 20-hydroxyecdysone were applied topically to the larvae 7. In 3 insect species with imaginal reproduction 8 11 20-hydroxyecdysone was found to have an almost identical effect on the ovary, i.e. to induce the organization of follicles and their separation. In order to find out whether the stage of follicle formation in the ovary of the H.pygmaea larva is characterized by an increase of ecdysteroids in situ, we measured the amounts of 20-hydroxyecdysone and ecdysone in the pedogenetically reproducing larvae throughout the life cycle, by using thin layer chromatography (TLC) in combination with a radioimmunoassay (RIA). Since fresh fungus fed to the larvae has an effect on the ovaries similar to that of 20-hydroxyecdysone on ovaries cultured in vitro (i.e. it stimulates follicle formation), we also examined the fungus for the presence of ecdysteroids using TLC and high pressure liquid chromatography (HPLC) combined with a RIA. (Cecidomyiidae, Dipt.) were kept under conditions whereby they reproduce by thelytokous (female producing) pedogenesis only 12. Material and methods. Larvae of Heteropeza pygmaeaWhen fed on fresh fungus (the Basidiomycete Peniophora albuta, cultured on agar-malt substrate), the larva mainly consists of a body wall filled with female offspring at the end of the reproductive cycle. Young female larvae were collected at the moment of hatching from their mother larvae and then either homogenized and stored for further treatment (see below) or placed on food. A heap of 150 larvae was placed in the center of a Petri dish with fresh (3-day-old) fungus and then kept in the dark at 25~ Gen...