Pars intermedia and pars distalis of the rat hypophysis were examined, following experimentally induced alterations in ACTH secretion. The concentration of ACTH in the pars inter-media is considerably greater than that in the pars distalis. At 1 month after adrenalectomy, the ACTH concentration in the pars distalis increased 3-fold, while that in the pars intermedia remained unchanged. Following chronic administration of cortisol, the ACTH concentration in the pars distalis fell to 27% of the control, while the fall in ACTH concentration in the pars intermedia was not significant. The immunoglobulin-peroxidase bridge technique, employing 17–39 ACTH antiserum, demonstrated the presence of ACTH-containing cells, both in the pars intermedia and in the pars distalis. Following adrenalectomy or chronic administration of cortisol, there was no change in the immunostaining or in the electron microscopic appearance of the cells of the pars intermedia. These findings suggest that although a relation between the pars intermedia and ACTH is established, this relation is different from that between the pars distalis and ACTH.