Rat thyroid tissue and three rat medullary thyroid carcinoma cell lines, 6-23, WE4/2, and CA77, have been examined for substance P (SP) and SP-like peptide expression. Analysis by combined HPLC and radioimmunoassay revealed the presence of SP in thyroid and 6-23 cell extracts. The presence of SP-encoding mRNAs was also detected in 6-23 cells by solution hybridization-nuclease protection analysis. SP-encoding mRNA expression was increased (fourfold) by maintaining the 6-23 cells in low serum (2%) for 4 or 10 days. The 6-23 cells also expressed other SP-like immunoreactive species, which were chromatographically and immunologically distinct from established tachykinin peptides. WE4/2 cells did not contain SP but did display SP-like immunoreactivity (SPLI), which migrated like the unidentified SPLI in 6-23 cells. CA77 cells did not contain SP or SP-encoding mRNA but did contain SPLI that migrated identically to the unidentified SPLI in the other cell lines. This novel SPLI was detected with an antiserum directed against the SP carboxyl terminus and to a lesser extent with an antiserum directed against the neurokinin A carboxyl terminus, but it showed minimal cross-reactivity using an antiserum directed against the midportion of SP. Treatment with 50 mM KCl resulted in secretion of this SPLI from CA77 cells. Gel filtration analysis demonstrated that this novel SPLI had an apparent molecular weight of approximately 1,000. These results are discussed in terms of cell lines that express tachykinin peptides and in terms of the molecular nature of the new SPLI detected in CA77 cells.