We have recently reported the establishment of 16 series of calcitonin-producing transplantable rat medullary thyroid carcinoma. In the present study, these tumor series have been evaluated for the presence of somatostatin-like immunoreactivity. Each of the series contained detectable levels of both peptides. Immunoreactive somatostatin varied from less than 1 ng/mg of protein to almost 500 ng/mg of protein. The range of immunoreactive calcitonin was 0.3 to 30 micrograms/mg of protein. Although somatostatin-like immunoreactivity was always less than that of calcitonin, the levels in certain series were as high as those found in neural or endocrine tissues used for in vitro studies of somatostatin elaboration. No significant correlation was found between tissue levels of these two peptides. Two tumor lines were generated by initiation of tumor growth with cells from primary monolayer cultures. Levels of both immunoreactive calcitonin and somatostatin significantly differed from those of the parent lines, which were maintained by serial passage of tissue fragments only. Plasma somatostatin-like immunoreactivity assessed in two tumor series with high (149 ng/mg of protein) and low (1.5 ng/mg of protein) tissue levels was 3100 and 50 pg/ml, respectively. Gel filtration chromatography of tissue and blood extracts showed a predominant peak (greater than 90%) of immunoreactive somatostatin eluting at the position of the native hormone. Three other peaks were resolved in the tissue extract with estimated molecular weights of 14,000, 8,700, and 5,000. The high level of somatostatin-like immunoreactivity and the presence of multiple large forms suggest that certain tumor lines will prove valuable for studies of somatostatin biosynthesis and secretion.