The relationship between hemolymph ecdysteroid titer, ring gland (RG) activity, and prothoracicotropic hormone (PTTH) activation of RG in vitro has been examined during the postfeeding larval, prepupal, and pupal stages of Sarcophaga bullata. Using the ecdysteroid radioimmunoassay (RIA), two significant peaks were recorded during the red spiracular stage and during the first few hours after the formation of the white prepupa and a third large peak 9 hr later. It is postulated that these increases in ecdysteroid titer are involved in the processes of pupariation, puparial tanning, and pupation, respectively. Ring glands isolated from Sarcophaga of known ages were incubated in vitro and the secreted ecdysone was quantified by RIA. Ring glands from early red spiracular stage larvae proved to be the most active and subsequent secretory activity of the RG oscillated every 4 hr with the oscillations gradually decreasing in amplitude. RG activity returned to a basal level 24 hr after formation of the white prepupa, about the time that the hemolymph ecdysteroid titer fell to its basal level. To demonstrate PTTH activity in vitro, brains from 3- to 4-hr prepupae were chosen to activate ring glands from postfeeding larvae. Using a graded series of dilutions of PTTH extract it was shown that a dose-response relationship could be obtained for Sarcophaga similar to that demonstrated or the Manduca sexta PTTH-prothoracic gland system. In Sarcophaga maximal activation resulted in a 10-fold increase in ecdysone synthesis and secretion by ring glands stimulated with 0.5 brain eq. Half-maximal stimulation was attained with 0.2 brain eq of PTTH extract.