We have reported previously the inhibitory effect of 4-thiouridine (4SU) on chloroplast development in greening radish cotyledons. In this paper we present the results on metabolism of 4SU in germinating radish seedlings. Radish seeds were germinated and grown with (treated) or without (untreated) 4SU in the dark for 4 days. RNA prepared from the treated cotyledons had an absorption peak near 330 nm ; however RNA from the untreated ones did not show any siginificant peak in this region. Selective modification of RNA from the treated cotyledons with hydrogen peroxide or hydroxylamine, which are known to react specifically with 4SU or 4SU moiety in RNA, resulted in the disappearance of the peak near 330 nm. About 15-20% of RNA from the treated cotyledons bound to mercurated cellulose and was eluted with mercaptoethanol. These results indicated that 4SU is metabolized to 4-thioUMP, 4-thioUDP and 4-thioUTP and then incorporated into RNA through the same pathways as is uridine in the germinating radish seedlings. We consider that the action of 4SU on chloroplast development may be through the depressed activity of translation or an abnormal translation due to the 4SUcontaining RNA or the inhibition of RNA synthesis by the 4SU moiety in developing plastids.