Injection of hemolymph from orange puparia or of CNS-extracts from larvae into red-spiracle larvae of Sarcophaga bullata accelerates the onset of pupariation in relation to retraction of the anterior segments and tanning. By the use of ammonium sulfate precipitation, heat treatment, ultracentrifugation, ultrafiltration, gel filtration, and electrophoresis, two proteinaceous factors were isolated and partially purified. One of these, called ARF (anterior retraction factor), accelerates retraction and has no tanning activity. The other, called PTF (puparium tanning factor), accelerates tanning and has no ARF activity. The ARF has a molecular weight of about 180,000, is heat labile, and was purified about 170 times. The PTF has a molecular weight of about 312,000, is heat stable, and was purified about 200 times. Similar factors were found in hemolymph and CNS-extracts, however, hemolymph was more active in ARF, and CNS in PTF. These factors are most probably not related to two other brain hormones, ecdysiotropin and bursicon, because of differences in molecular weights, electrophoretic mobilities, and distribution. Based on the similarities in the nature of the ARF and PTF in hemolymph and CNS it is suggested that they originate in the CNS, are stored in peripheral nerve endings, and released through the action of ecdysone into the hemolymph during pupariation. The factors have no effect in the precritical (36 hours before pupariation) and early post-critical (10-12 hours before pupariation) stage larvae. They are functional only in a red-spiracle larva (2-4 hours before pupariation). This suggests that they cannot replace ecdysone in effecting pupariation, and that they require ecdysone-dependent enzyme proteins or other co-factors which become available only in the red-spiracle larva. The role of such substances may be to alter the permeability of target cell walls in order to facilitate the entry of ARF and PTF.