Spasmolytic polypeptide (SP) and intestinal trefoil factor (ITF) are trefoil peptides expressed by gut mucus cells. Using specific antisera we have quantified and characterized the molecular forms and distribution of these peptides in the rat gut. SP predominates in the gastric antrum as a 12 kDa form. ITF (7 kDa) is highly expressed throughout the small intestine. Both peptides are distributed in the apical secretory compartment of antral mucus cells (SP) and goblet cells (ITF), and on the lumenal surface. This study quantifies SP and ITF for the first time, and confirms them as major secretory products of the rat gut.