The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with Kd of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with Kd 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with Kd 0.42 was identified as rat alpha-CGRP as was the peak with Kd 0.53. The peak with Kd 0.68 was identified as 19-37 rat alpha-CGRP and the peak with Kd 0.85 as 28-37 rat alpha-CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated alpha-CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat beta-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.