Irreversible loss of the ability to develop chloroplasts in Euglena gracilis may develop following transfer from organic medium to defined medium. Requirements for the loss include the absence of light and a temperature of 30 C (the optimal temperature for multiplication) although neither darkness alone nor this temperature alone serves as the bleaching agent. The extent of bleaching of a population can approach 100% but depends heavily on the following conditions: the pH of the defined medium and its phosphate content, the age of the parent culture at transfer, and the length of time spent in the defined medium before cell divisions are permitted. Bleaching is not due to loss of nonreplicating proplastids through "dilution out" as cells divide but appears to be a more direct inactivation of chloroplast differentiation from proplastids.