There has been no direct evidence to establish that the prothoracic glands are responsible for ecdysone synthesis. Our improved culture system, in which the isolated prothoracic glands of the Bombyx silkworm were cultivated in hemolymph medium under a continuous supply of excess oxygen, led to first successful result. A typical set of data (Table 1) reveals that a considerable amount of hormonal activity is found in the medium. The hormonal fraction was extracted from the culture medium and applied to a TLC plate. The results revealed that all the hormonal activity was located in a distinct region corresponding to α or β-ecdysone. The ecdysone fraction was submitted to high pressure liquid chromatography. Only fraction 8 (Fig. 5) having the same retention time with α-ecdysone had the hormonal activity On the other hand, a specific gas phase analysis of zooecdysones using mass fragmentography of trimethylsilyl derivative was developed. Quantitative estimation of 10^<-10> g level of α and β-ecdysone can be obtained by focusing at m/e 564 and 561, respectively. Analysis of the fraction 8 by this technique clarified that the hormone obtained from organ culture is α-ecdysone.