The distribution of those cells in the anterior pituitary gland of the rat which stain immunohistochemically with rabbit anti-human calcitonin serum has been examined. Immunoreactive cells were confined primarily to the ventral surface of the gland and possessed both a distribution and morphology distinct from the corticotrophs. Staining of serial thin sections with rabbit anti-rat TSH-subunit serum resulted in an immunoreaction in those cells that stained for calcitonin. However, not all the thyrotrophs gave a positive immunoreaction for calcitonin. It is concluded from this study that it is inappropriate to attribute calcitonin as being part of the 31K-dalton precursor for adrenocorticotropin, a hypothesis that was proposed earlier. In the immunohistochemical reaction with anti-calcitonin serum, it was found that relatively high concentrations of antigen (500 micrograms/ml) were required in absorption experiments in order to inhibit staining. Furthermore, the staining of thyrotrophs with the anti-calcitonin serum was inhibited after preadsorption of the primary antiserum with excess rat beta-TSH (1000 micrograms/ml). Because of these immunochemical characteristics, it is questionable whether the calcitonin-like material observed in the rat pituitary gland is chemically identical to that of thyroidal calcitonin.