Autocrine activation of DNA synthesis in prothoracic gland cells in last instar larvae of the silkworm, Bombyx mori, was studied using both a long-term in vitro organ culture system and immunocytochemical labeling with 5-bromo-2'-deoxyuridine (BrdU). When prothoracic glands were incubated in a small volume of culture medium (10 microl/gland), the numbers of DNA-synthesizing cells per gland increased significantly, and DNA synthesis was stimulated less by hemolymph, as compared with glands incubated in a large volume (50 microl/gland). Moreover, glands cultured in groups (6 glands per group in a 50-microl drop) also resulted in much higher levels of DNA synthesis than those cultured individually in a 50-microl drop. The mechanism by which alternation of the volume of the incubation medium results in changes in the levels of DNA synthesis was further examined. When prothoracic glands were incubated in medium (50-microl drop per gland) that was preconditioned with glands (in a 10-microl drop individually), a dramatic increase in DNA synthesis activity was also observed, indicating that prothoracic glands may release a factor that stimulates their own DNA synthesis. The growth-promoting factor was further characterized and it was found that the factor is heat stable, and its molecular weight was estimated to be between 1,000 and 3,000 Da. Moreover, the factor also stimulated corpus allatum cell DNA synthesis in vitro. Injection of concentrated putative growth-promoting factor into day 4 last instar-ligated larvae greatly increased cell DNA synthesis of the prothoracic glands, indicating the in vivo function of the present autocrine factor.