We have reported that both an extract of rat hypothalamus-stalk-median eminence (HE) and 5-hydroxytryptamine (5-HT) will stimulate the release of ACTH biological activity from acutely, non-enzymically dispersed rat pars intermedia (PI) cells. We have also found that the ACTH activity within the PI cells is composed of 4 chromatographically separable entities, but only a small proportion of the total ACTH activity co-elutes with ACTH1-39. In the present study we subjected the media, after incubation of PI cells with either HE or 5-HT, to sequential chromatography on Sephadex G-50 F and G-100 SF. We found the same 4 ACTH-like moieties as in the PI cell extracts. Only the release of the 2 earliest eluting components, presumably of much larger molecular dimensions than ACTH1-39, was enhanced by either HE or 5-HT. There was no augmented release of the ACTH component having molecular dimensions similar to ACTH1"39. Thus, in our in vitro system, the ACTH biological activity released from PI cells is not the size of ACTH1-39, but is composed of at least 2 much larger molecules which possess ACTH (as well as some MSH) biological activity.