We studied the distribution of transcripts encoding the cytoplasmic domain of the membrane-anchored precursor epidermal growth factor (proEGFcyt) and a novel cytoplasmic proEGF splice isoform with a deleted exon 23 and an out-of-frame fusion of exon 24 (proEGFdel23) in human normal and neoplastic thyroid tissues. In papillary thyroid carcinoma (PTC), coexpression of transcripts encoding for both proEGFcyt and proEGFdel23 correlated with poor differentiation of PTC. To determine potential roles of the cytoplasmic proEGF domain in human thyroid cells, we generated stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 overexpressing the normal cytoplasmic domain proEGFcyt, a truncated proEGFcyt composed of the peptide sequence encoded by exons 22 and 23 (proEGF22.23) and proEGFdel23. The proEGFcyt and proEGF22.23 transfectants displayed significantly reduced proliferation rates, an enlarged cellular phenotype, and alterations in the distribution and post-translational modification of the microtubular system. These transfectants also displayed increased production of microtubule-associated proteins 1b and 2c, which was absent in FTC-133-proEGFdel23 or FTC-133-empty plasmid transfectants. This is the first evidence of an involvement of proEGF cytoplasmic domain in microtubular stability in the human thyroid carcinoma cell line FTC-133 and may suggest a specific role for the cytoplasmic domain of membrane-anchored proEGF, particularly exon 23, in thyroid carcinoma. The up-regulation of proEGFdel23 in poorly differentiated PTC and the exclusive detection of both proEGF isoforms in undifferentiated thyroid carcinoma may indicate an involvement of this novel truncated proEGFdel23 cytoplasmic domain during dedifferentiation processes of human thyroid cells.