1. The effect of ligation on pupariation in the front or hind parts of larvae of four species of flies, Calliphora erythrocephala, Phormia regina, Sarcophaga bullata, and S. argyrostoma was investigated. Ligation causes effects of delay or inhibition of pupariation which are very differently expressed in the four species. A large proportion of pre- or postcritically ligated specimens of P. regina and S. bullata altogether fail to pupariate in the anterior part. This makes these species unsuitable test subjects for the pupariation test for ecdysone. 2. Test abdomens of C. erythrocephala required significantly less ecdysone for a given pupariation effect when also injected with a CNS-extract. Tanning was also considerably accelerated in this case. 3. The value of the pupariation unit of ecdysone is influenced by a number of factors, such as age at the time of ligation, the waiting period between ligation and injection, the dilution effect of the solvent, and the simultaneous action of a neurohormone. The requirements for natural ecdysone in normal larvae at the time of pupariation are probably substantially lower than the values which have been determined by others with test abdomens and the use of synthetic ecdysones. 4. In confirmation of older data, and contrary to recent claims, tanning was induced in test abdomens of the larvae of C. erythrocephala, P. regina, and S. argyrostoma by the injection of hemolymph from pupariating larvae. Calliphora blood induced tanning in specimens of S. argyrostoma, and vice versa. The conclusions are drawn that differences between the different species in the action of ecdysone are of a quantitative rather than qualitative nature.