The prothoracic gland (moulting gland) of Periplaneta americana, the main source of ecdysteroids, was found to secrete proteins besides ecdysteroids. The course of ecdysone and protein secretion during the last larval instar was determined under in vitro conditions. Ecdysteroid production in the prothoracic gland as measured under in vitro conditions starts to increase to a first maximum between the fifteenth and twentieth days in the last larval instar. The main peak of ecdysteroid production is between the twenty-fifth and thirtieth and/or thirty-first days (i.e., at the end of the instar). Under the influence of actinomycin D, or puromycin (10 -5 M) in vitro ecdysteroid release by the prothoracic gland is decreased by about 80%. On the fifth day of the last larval instar the glands show the lowest level of total protein content (5 Β΅g per gland). Between the sixteenth and twentieth days, the protein content increases to about 9 Β΅g, remaining at 7-9 Β΅g per gland up to the end of the larval stadium. Prothoracic glands secrete proteins into the culture medium. Between the tenth and sixteenth days, protein secretion increases to a maximum level of about 8 Β΅g per gland during a 4 h incubation interval. After the eighteenth day, the level decreases to 2-3 Β΅g per incubation interval, with a transient significant increase on days 20 and 24 to about 5 Β΅g. Some of the synthesized proteins serve a specific function in the gland cells, while some others seem to be synthesized for release into the hemolymph. Release of proteins after the tenth day of the larval stadium has no correlation to ecdysteroid release. Proteins of this period obviously have a function independent of ecdysone, perhaps as mediators. Arch.