Using a radioimmunoassay specific for the carboxyl terminus of β-endorphin-(1–9) large amounts of β-endorphin-(1–9)-immunoreactive material was detected in the human pituitary. The major peak of immunoreactivity was purified and characterized by fast atom bombardment-mass spectrometry and Edman degradation sequencing as authentic β-endorphin-(1–9). In the rat pituitary the highest concentration of β-endorphin-(1–9) immunoreactivity was in the posterior neurointermediate lobe. This material was identified as N-acetyl β-endorphin-(1–9) by multiple radioimmunoassays, gel chromatography, and reversed-phase high-performance liquid chromatography. Control experiments determined that β-endorphin-(1–9) was not formed postmortem or during the extraction procedure. These studies suggest that single lysine residues, similar to single arginine residues, are potential sites of posttranslational processing.