Strains of rat medullary thyroid carcinoma cells were established by two different techniques which were designed to ensure that clonal populations originated from single cells. The clonal strains (44-3C1 and 6-23C6) secreted both immunoreactive calcitonin (CT) and neurotensin (NT) and had growth characteristics that were similar to those of the parent cell lines. Both the 44-3C1 strain and the parent 44-2 cell line consistently produced more CT and NT than the 6-23C6 clonal strain or the parent 6-23 cell line. Peptide secretion in these clonal strains was stimulated by calcium and norepinephrine. Both clonal strains, similar to the parent lines, produced 4 to 12 times more NT than CT. Immunohistochemical studies showed that all cells in both clonal strains stained positively for CT. NT was also present in all cells from both strains with approximately 2% of the cells showing intense staining for this peptide. Ultrastructurally, the cells contained membrane-bound secretory granules which had a mean diameter of 95 nm. Secretory granules were relatively numerous in only 2% of the cells where they tended to be concentrated in cell processes. These studies demonstrate that single rat medullary thyroid carcinoma cells produce both CT and NT and that these strains are useful models for studies of NT and CT biosynthesis and for investigations of the regulation of secretion of two peptides from a single cell type.