The extractability of neurotensin (NT) from porcine ileal mucosa was studied by comparison of eight extraction procedures. Tissue content of neurotensin-like immunoreactivity was quantitated and characterized by sequence-specific radioimmunoassays and gel filtration chromatography. Homogenization prior to boiling in extraction solvent produced higher levels of the intact peptide than the reverse procedure. N-terminal immunoreactivity was not influenced by the sequence of these steps. Tissue levels of intact NT were highest after extraction with 2.0 M acetic acid (mean 79.1 pmol/g, N = 6) and lowest with distilled water (mean 6.5 pmol/g, N = 6). The opposite was the case with levels of N-terminal immunoreactivity (mean 55.2 pmol/g and 105.7 pmol/g respectively, N = 6). Recovery experiments with addition of synthetic NT 1-13 and the N-terminal fragment NT 1-8 indicated that these differences could be explained by differences in recovery of intact NT and N-terminal immunoreactive components in tissue. Gel chromatography confirmed that in acetic acid almost only the intact peptide was extracted from ileal mucosa, and showed that after extraction in water or phosphate buffer several N-terminal components were present. The results suggest that a molecular heterogeneity may be present in ileal tissue. If this concept is supported by further studies differential extraction procedures may be needed in the future.